HPV Detection and Quantification by Real-Time Multiplex Application

ABSTRACT

The present invention relates to amplification primers and detection probes, which are useful for the detection of human papillomaviruses (HPV), and more particularly of HPV, which can be oncogenic for the mucosal epithelia. The amplification and detection systems provided by the present invention are group-targeted systems, namely A5-, A6- A7-, and A9-targeted systems. The amplification and detection systems of the invention allow for an amplification of HPV in multiplex as well as for a real-time detection, whereby at least the thirteen HR HPV can be detected in a single-tube assay. The invention further allows for a reliable quantitation of HPV viral loads in real-time multiplex amplification.

FIELD

The present invention relates to the detection of human papillomaviruses(HPV), more particularly of HPV, which have a tropism for the mucosalepithelia (mucosal-type HPV), still more particularly of HPV, which canbe oncogenic for the mucosal epithelia. The present invention providesamplification primers and detection probes, which are useful therefor,as well as reference template sequences suitable for designing andbuilding such primers and probes.

BACKGROUND OF THE INVENTION

Human papillomavirus (HPV) contains a circular double-stranded DNAgenome of about 7,900 bp, which is organized into three main regions,i.e.:

-   -   an early coding region containing genes E1, E2, E4, E5, E6 and        E7, which are involved in viral replication and in neoplastic        transformation,    -   a late coding region, containing genes L1 and L2, which code for        viral capside proteins,    -   a non-coding regulatory region, which is referred to as LRC        (Long Control Region), which is located between the E genes and        the L genes.

HPV constitute a group of viruses, which are associated with benign andmalignant lesions of cutaneous or mucosal epithelia. To date, more than100 different HPV types have been identified.

More than 40 HPV types belonging to the mucosal group have been detectedin the anogenital mucosa.

HPV is the major risk factor in the development of squamousintraepithelial lesions (SILs), which are classified as low grade (LSIL)or high grade (HSIL) in severity.

HPV may induce cervical intraepithelial neoplasia (CIN), ranging frombenign lesions (CIN1), such as condylomata acuminata, throughpre-cancerous lesions (CIN2 to CIN3), up to in situ carcinoma andinvasive cancer.

It is now established that HPV is directly involved in cervicalcarcinogenesis. Detecting HPV is essential to the prognosis of CIN andcervical cancer.

Early and precise detection of HPV is the key factor for recovery fromcervical cancer.

It has also been shown that an increased HPV viral load within acervical smear to specimen is associated with an increased risk of CIN3and of cervical carcinomas.

A number of oncogenic HPV genotypes that infect the anogenital tracthave been classified as potentially high risk HPV genotypes (HR HPV),based on their occurrence or prevalence in cervical carcinomas. Thepresence, persistence and/or re-occurrence of HR HPV is a bad prognosticindicator. So far, thirteen HPV types are said to be HR HPV, namely HPV56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59, 45 and 18. Those HR HPV,which have the highest prevalence, are HPV types 33, 31, 16, 45 and 18.

Other oncogenic HPV are considered to be Low Risk HPV (LR HPV), e.g.,HPV2, HPV3, HPV6, HPV11, HPV13, HPV32, HPV40, HPV42, HPV43, HPV44,HPV57.

The clinical classification of HPV types into either the HR or the LRgroup might evolve, or slightly diverge from one author to another, asthe classification of a given HPV into the LR group only stands for aslong as this HPV type is not found to be associated with a cervicalcarcinoma.

For example, it is now contemplated that HPV53 and HPV66 probably are HRHPV (van Ham et al. 2005, J. Clin. Microbiol. Vol. 43, n^(o) 6, p.2662-2667). Hence, the initial group of thirteen HR HPV might furtherincrease to a number of at least 15 HPV types.

Other HPV have been described as oncogenic HPV, but without anydefinitive settlement on the issue of their HR or LR status, such as isthe case for HPV67, HPV82, HPV85. Appropriate detection means arerequired to analyze their oncogenicity.

New, or yet unidentified, mucosal oncogenic HPV types may further arise.Furthermore, HPV multi-infection, involving several types of HPV, is acommon situation: multi-infection is thought to account for about 20% ofthe HPV infection cases. An HPV multi-infection case may involve HPVtypes, which all are oncogenic HPV, or which comprise at least oneoncogenic HPV and at least one non-oncogenic HPV. An HPV multi-infectioncase may involve HPV types, which all are mucosal HPV types, or mayinvolve at least one mucosal type and at least one cutaneous type.

Also, co-infection may also occur, which involves at least one HPV andat least one virus other than HPV, e.g., a co-infection with at leastone HPV, and at least one HIV.

Such multi- and/or co-infection situations render accurate HPV detectionmuch more difficult. HPV primers and probes, which are suitable for thedetection of mucosal oncogenic HPV, have been disclosed in prior art.

The first techniques that were developed involved type-specific probes,which were designed to detect oncogenic HPV by direct hybridization of atype-specific probe to a non-amplified HPV genome, e.g., by Southernblotting or dot blotting.

Signal-amplified tests have then been developed, such as the HybridCapture test (HC2®) of Digene Corporation, Gaithersburg, Md., USA. TheHC2® test has been approved by the FDA, and is at present time thereference test for clinical diagnosis.

The HC2® system is a liquid phase microplate system using DNA/RNAhybridization assay, which does not comprise any target amplification:viral DNA hybridizes in liquid phase to a RNA probe which targets the 13HR HPV, the hybrids thus formed being detected by anti-DNA/RNAantibodies and visualized by chemoluminescence.

The HC2® test is a sensitive assay. It however is only of qualitativevalue. Viral loads assessed by the HC2® test does not increase withincreasing grade of SIL, and are not sufficiently reliable in case ofmultiple HPV infections. Hence, the HC2® test is not a quantitativeassay.

As the HC2® test does not reflect the viral load initially contained inthe analyzed sample, it is recommended to combine it with classiccytology, to distinguish the cases with high grade lesions from thosewithout high grade lesions.

Amplification methods have then been developed, wherein HPV target(s)is(are) amplified by at least one primer pair, the amplicon thusproduced being detected either by this (labelled) primer pair or by aprobe.

Such prior art primers have first been designed as general consensusprimers, which are intended for amplifying several HPV, usually severalof the thirteen HR HPV, as well as other HPV (oncogenic LR, andsometimes also non-oncogenic HPV).

Such consensus primers are also referred to as “universal” primers.These consensus primers target conserved regions in the HPV L1 gene(e.g., the MY09/MY11/HMB01 primers, the GP5+/GP6+ primers, thePGMY09/PGMY11 primers, and the SPF1/SPF2 or SPF10 primers), or the E1ORF region (e.g., the CPIIG/CPI primers described in Tieben et al. 1993,J. Virol. Methods 42:265-279).

To render consensus PCR applicable to clinical diagnosis, HPV probeshave been developed to detect and type HPV amplicons generated byconsensus primer sets. Detection of the HPV amplicons generated byconsensus primers is usually performed by a reverse hybridization lineblot assay, or by calorimetric microtiter plate-based enzymeimmunoassay.

Illustrative of such consensus PCR methods are the INNO-LiPA HPV test(Innogenetics, Gent, Belgium), and the Amplicor HPV test (RocheMolecular Systems, Branchburg, N.J., USA).

The INNO-LiPA HPV test is a reverse hybridization line probe assay, theprototype research version of which has been described in Kleter et al.,1999 (Journal of Clinical Microbiology, vol. 37, n^(o) 8, p. 2508-2517),and Kleter et al. 1998 (American Journal of Pathology, vol. 153, n^(o)6, p. 1731-1739),

Briefly, a PCR primer set is used to generate a short PCR fragment (SPFPCR) of 65-bp from the L1 open reading frame. The prototype researchINNO-LiPA primer set consists of 10 biotinylated primers (referred to asthe SPF10 primer set), namely the six primers of the SPF1/2 system(described in Kleter et al. 1998), and four additional primers (MY09/11and GP5+/6+).

The SPF10 amplimers are denatured, and incubated under hybridizationconditions with poly(dT)-tailed type-specific oligonucleotide probes,which are immobilized as parallel lines on nitrocellulose membranestrips. The probe strips are then washed out for detection of theretained hybrids.

The INNO-LiPA HPV test allows the detection of at least 25 HPV genotypes(the 13 HR HPV, i.e., HPV 56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59,45, 18, as well as other HPV, e.g., HPV 6, 11, 34, 40, 42, 43, 44, 53,66, 70, and 74). It is a genotyping line probe assay, and is ofqualitative value. The INNO-LiPA HPV test is not a quantitative assay.

The Amplicor HPV test uses amplification of target HPV DNA by PCRfollowed by nucleic acid hybridization for the detection of the thirteenHR HPV. The Amplicor HPV test amplifies a sequence of about 165 bpwithin the L1 region. The primer sets consist of 12 primers, which havebeen designed as general consensus primers, to amplify the initial groupof 13 HR HPV. After amplification and denaturation, the amplified HPVsequences are distributed in a microwell plate, and incubated with L1capture probes, the hybrids being detected and visualized bycolorimetric enzyme immunoassay (avidin-horseradish peroxidaseconjugate). The Amplicor HPV test has been reported as being of higheranalytical specificity, compared to the HC2® test (less false negativeresults, see Poljak et al. 2005, Acta Dermatoven APA, vol. 14, n^(o) 4,p. 147-152).

The Amplicor HPV test is sensitive, but its HPV spectrum is restrictedto those 13 HPV, which have been initially considered as being the HRHPV. For example, the Amplicor HPV test does not detect HPV66 and HPV53,which are now thought to be HR HPV. In other words, the Amplicor test isnot designed to be adaptive to any change or evolution in HPVclassification or knowledge.

Furthermore, the Amplicor HPV test is not a quantitative assay.

These line blot or microwell-based prior art techniques use consensusHPV primers, i.e., primers which result from sequence alignment of apre-determined set of selected oncogenic HPV, and from the determinationof those consensus sequences, which have a sufficient similarity oridentity score with all of the selected HPV, to hybridize to all ofthem. Consensus primers are thus designed to amplify a predeterminedsub-set of oncogenic HPV, and may not succeed in amplifying otheroncogenic HPV (such as HR new corners, or non-HR oncogenic HPV).

Such a consensus approach is restricted by the possibility ofdetermining primer sequences, which would still sufficiently hybridizeto the ever increasing and ever evolving desired targets.

If one or several new oncogenic HPV strain(s) appear, such prior artconsensus primers might give false negative results.

Also, none of the prior art line blot or microwell-based techniques isof a quantitative nature, whereas recent findings show that the HPV copynumber accounts for the phase and/or severity of the disease, and/orhave a diagnostic and/or prognostic value in the field of oncogeny.

Absence of quantitative performance limits the spectrum of clinicalapplicability, as such tests cannot give information on the actualcancer risk level, or on the actual cancer grade.

Moreover, according to these prior art consensus line blot ormicrowell-based techniques, the detection step is an additional andtedious step, which has to be performed as a separated step afteramplification has occurred.

Real-time PCR amplification techniques have recently been developed forthe detection of HPV16 or HPV18 (Hesselink et al. 2005, Journal ofClinical Microbiology, vol. 43, n^(o) 9, p. 4868-4871; Gravitt et al.2003, Cancer Epidemiology, Biomarkers & Prevention, vol. 12, p.477-484).

These real-time PCR either are based on FRET (LightCycler), or useTaqMan probes (Applied Biosystems).

Compared to prior art line blot or microwell-based techniques, suchreal-time PCR have the advantage of combining amplification anddetection in one single step, and of opening the way to quantification.

For example, van Duin et al. 2002 (Int. J. Cancer, vol. 98, n^(o) 4, p.590-595) describes a quantitative real-time PCR assay for the detectionof HPV16.

Prior art real-time PCR protocols however are type-specific PCRprotocols, which are limited to the detection of only one HPV peramplification run, and more particularly to the sole detection of HPV16or HPV18. They thus represent valuable research tool, but have a verylimited clinical applicability.

Attempts have been made to develop multiplex real-time PCR amplificationof HPV. These attempts are however limited to duplex or triplexreal-time PCR for the detection of HPV16, HPV18, HPV45. For example,Szuhai et al. 2001 (American Journal of Pathology, 159(5): 1651-1660)disclose seven type-specific to molecular beacons, which are said to betype-specific molecular beacons, namely five HR HPV molecular beacons(HPV16, 18, 31, 33, 45) and two LR HPV molecular beacons (HPV6, 11); seetable 1 of Szuhai et al. These molecular beacons are described as beinguseful for the detection of amplicons generated by the CPI/CPIIG“universal” primers. Multiplex attempts are disclosed in Szuhai et al.,but are limited to duplex or triplex assays (HPV16, HPV18, HPV45). Theauthors explicitly indicate that “although the multiplexing capacity ofmolecular beacon PCR is higher than three, it is unlikely that it willapproach the number of different HPV genotypes” (see page 1656,right-hand column, second paragraph). For this reason, the authors cameto the conclusion that type-specific molecular beacons cannot by theirown solve the problem of HPV clinical diagnosis, and that they shall beused in combination with a general pre-screening HPV detection method,to arrive at a two-step HPV detection and genotyping strategy (see e.g.,FIG. 6 of Szuhai et al.), wherein type-specific HPV molecular beacon PCRis disclosed to be used in combination with a SybrGreen general primerPCR pre-screening.

Hence, to the best of the inventors' knowledge, prior art does neitherdescribe nor suggest any real-time amplification technique that could beworked in multiplex, whilst retaining the required HPV detectionspecificity, which would allow to cover at least the 13 HR HPV in asingle step (amplification+detection) run. Furthermore, to the best ofthe inventors' knowledge, prior art does not describe any quantitativereal-time HPV amplification technique, which would allow to cover atleast the five most common HR HPV (namely, HPV16, 18, 45, 31 and 33),preferably at least the 13 HR HPV, more preferably at least the 13 HRHPV as well as five other oncogenic HPV, in a single step(amplification+detection) run, and which would be quantitative, evenwhen implemented in multiplex.

ABSTRACT OF THE INVENTION

The present invention relates to the detection of HPV by amplification,more particularly of mucosal-type HPV, still more particularly of HPV,which can be oncogenic for the mucosal epithelia.

The present invention allows the detection of at least the five mostcommon HR HPV (HPV16, 18, 45, 31, 33), preferably at least 7 HR HPV,still preferably the five most common HR HPV as well as at least twoother HR HPV, advantageously at least two other HR HPV belonging togroups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), morepreferably at least the 13 HR HPV (HPV 56, 51, 58, 33, 52, 35, 31, 16,68, 39, 59, 45, 18), most preferably at least the 13 HR HPV and fiveother oncogenic HPV (HPV66, 53, 82, 67, 85).

The present invention relates to amplification primer systems, and todetection probe systems, as well as to the amplification-detectionsystems (i.e. real-time amplification systems), which result from thecombination of at least one amplification primer system of the inventionand at least one detection probe system of the invention.

The present invention also relates to reference template HPV sequences,which are suitable for the production of amplification primers anddetection probes of the invention, as well as to amplicons obtainable byamplification of an HPV nucleic acid with at least one amplificationprimer system of the invention, and optionally by detection with atleast one detection probe system of the invention.

The present invention further relates to the biological andpharmaceutical applications thereof, more particularly to the diagnosticand prognostic applications thereof, notably in the field of CIN andcervical cancer.

The HPV amplification method of the invention is based on an approach ofHPV tropism and oncogenicity, which is completely different from, andcompletely innovative compared to prior art approaches: contrary to theglobal consensus approaches, or to the type-specific approaches, whichprior art methods have up to now followed, the present inventors havedesigned and built an approach, which is an HPV group-based approach(see the phylogenetic tree shown in FIG. 1, which has been built by thepresent inventors). According to the inventors' HPV group-basedapproach, amplification primer systems and detection probe systems areprovided for each HPV group that comprises at least one HPV type capableof being oncogenic for the mucosal epithelia, namely at least for eachof groups A6, A5, A9 and A7.

Indeed, the present inventors have selected appropriate targets withineach of said HPV groups, which are suitable for the production ofprimers and probes covering

at least the five most common HR HPV (namely, HPV16, 18, 45, 31 and 33),preferably at least 7 HR HPV, still preferably the five most common HRHPV as well as at least two other HR HPV, advantageously at least twoother HR HPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31,16, 45, 18), more preferably at least the 13 HR HPV, most preferably atleast the 13 HR HPV and five other oncogenic HPV (HPV66, 53, 82, 67,85), in a single step (amplification+detection) run.

The selected targets of the present invention are reference templatesequences, which allow designing and building primers (hybridizing toone end of said selected targets, or to the complementary sequencethereof), as well as amplicon-annealing probes, which allow to coversaid HPV in a single step (amplification+detection) run.

The amplification primer systems of the present invention are targetedto the HPV of group A6 or A5 or A9 or A7, and are intended to amplify asmany HPV types belonging to one of these groups as possible.

The detection probe systems of the present invention allow the detectionof one or several of the amplicon(s) obtainable by amplification of agiven HPV by an amplification system of the present invention. They aretargeted to group(s) A6 and/or A5 and/or A9 and/or A7, and areespecially adapted to implementation in real-time with saidamplification primer systems.

The detection probe systems of the invention comprise probes, whichallow for the general detection of at least one HPV that belongs to theHPV set formed by groups A6 and A5 and A9 and A7, as well as moreprecise detections, such as:

-   -   the detection of at least one HPV that belongs to group A6 or A5        or A9 or A7, or    -   the detection of at least one HPV that belongs to a sub-set of        group A6 HPV, or of group A5 HPV, or of group A9 HPV, or of        group A7 HPV, or    -   the detection of at least one particular HPV type.

The present invention thereby provides a great flexibility of precisionlevels for the detection of HPV. Such flexibility has, to the best ofthe inventors' knowledge, never has been previously attained.

The present invention further provides A6- and/or A5- and/or A9- and/orA7-targeted systems, resulting from the combination of at least oneamplification primer system of the invention and at least one detectionprobe system of the invention.

The present invention more particularly provides A6- or A5- or A9- orA7-targeted systems, which comprise at least one amplification primersystem of the invention and at least one detection probe system of theinvention, wherein said at least one amplification primer system andsaid at least one detection probe system are targeted to the same group,i.e., A6 or A5 or A9 or A7.

Most of the amplification primer and/or detection probe systems of thepresent invention comprise more than two primers and/or more than oneprobe. Hence, most of the amplification primer and/or detection probesystems of the invention, and notably the A9-targeted systems, and theA7-targeted systems, already are by themselves multiplex systems.

According to a very advantageous feature of the present invention, thegroup-targeted systems of the present invention are suitable for usetogether in a single-tube amplification, i.e., the present inventionallows for implementation of at least one A6-targeted system, and atleast one A5-targeted system, and at least one A9-targeted system, andat least one A7-targeted system, together in a single-tube assay,thereby resulting in what could be called a multi-multiplex, i.e., a“megaplex” amplification and/or detection: see e.g., in the examplesbelow, illustrative “megaplex” involving 17 primers and 12 probes, whichare capable of amplifying and detecting seventeen oncogenic mucosal-typeHPV in a single-tube assay, without any significant loss in specificity,and without any significant loss in sensitivity.

Hence, the amplification primer systems and detection probe systems arespecifically adapted to real-time multiplex amplification.

To the best of the inventors' knowledge, there is no prior art method,which would allow for a real-time multiplex amplification of at leastthe five most common HR HPV, preferably at least 7 HR HPV, stillpreferably the five most common HR HPV as well as at least two other HRHPV, advantageously at least two other HR HPV belonging to groups A6and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), more preferably atleast the 13 HR HPV.

Advantageously, the present invention allows for the detection of atleast 18 oncogenic mucosal-type HPV in a single-tube assay, namely the13 HR HPV, as well as five other oncogenic HPV (HPV66, 53, 82, 67, 85).

A further advantageous aspect of the present invention is that it isespecially adapted to HPV viral load quantification. The HPV method ofthe invention is able to remain specific and quantitative, even whenimplemented in a single-tube multiplex.

The amplification primer systems and detection probe systems arespecifically adapted to real-time quantitative multiplex amplification,and retain this capacity even when implemented in a “megaplex” modecomprising at least one A6-targeted system, and at least one A5-targetedsystem, and at least one A9-targeted system, and at least oneA7-targeted system, together in a single-tube assay.

To the best of the inventors' knowledge, there is no prior art method,which would allow for a real-time quantitative multiplex amplificationof said at least five most common HR HPV, preferably at least 7 HR HPV,still preferably the five most common HR HPV as well as at least twoother HR HPV, advantageously at least two other HR HPV belonging togroups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), morepreferably at least the 13 HR HPV, more preferably at least the 13 HRHPV and five other oncogenic HPV (HPV66, 53, 82, 67, 85).

The amplification primer systems and the detection probe systems of theinvention all share the special technical feature of being designed andbuilt according to a group-based approach of HPV oncogenicity, and ofbeing suitable for implementation together in a the same assay tube.

More particularly, they enable a real-time “megaplex” implementationcovering at least the five most common HR HPV types, preferably at least7 HR HPV (e.g., HPV 56, 51, 33, 31, 16, 45, 18), still preferably thefive most common HR HPV as well as at least two other HR HPV,advantageously at least two other HR HPV belonging to groups A6 and/orA5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), more preferably at least the13 HR HPV, more preferably at least 18 oncogenic mucosal-type HPV (i.e.,at least the 13 HR HPV, and five other oncogenic HPV, e.g., HPV66, 53,82, 67, 85), in a single-tube assay, without any significant loss in thequalitative accuracy of the HPV detection.

The amplification primer systems and the detection probe systems of theinvention further show levels of specificity, Ct and sensitivity, whichare sufficiently homogeneous to allow for a real-time “megaplex”amplification, which is of quantitative value.

The group-based approach of the present invention further providesflexibility to the amplification and/or detection systems, as theirintrinsic design is likely to make them suitable for detection of anyoncogenic HPV “new corner” that may arise by mutagenesis.

The group-based approach of the present invention also confersflexibility in the use for clinical diagnosis: depending on the choiceof probe system(s) that is made by the user, the precision level in HPVdetection can range from a general response indicating the detection ofat least one HPV belonging to the set formed by groups A6 and A5 and A9and A7, to the very precise response indicating the detection of atleast one particular HPV type. To the best of the inventors' knowledge,such flexibility has up to now never been attained.

The group-based approach of the present invention further is suitablefor providing accuracy in case of multi- and/or co-infections.

It is believed that such an achievement represents a technologicalbreakthrough, compared to prior art HPV detection systems.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: phylogenic tree of the present invention;

FIGS. 2A and 2B: schematic presentation of the amplification targets forHPV groups A6 (HPV56) and A5 (HPV51) (FIG. 2A), and for groups A9(HPV16) and A7 (HPV18) (FIG. 2B);

FIG. 3: reprint of NCBI_(—)001594.1, sequence of HPV56 (reference HPVfor group A6); SEQ ID NO:420;

FIG. 4: reprint of NCBI_(—)001533.1, sequence of HPV51 (reference HPVfor group A5); SEQ ID NO:421;

FIG. 5: reprint of NCBI_(—)001526.1, sequence of HPV16 (reference HPVfor group A9); SEQ ID NO:422;

FIG. 6: reprint of NCBI_(—)001357.1, sequence of HPV18 (reference HPVfor group A7); SEQ ID NO:423;

FIG. 7: convention for positions, which is followed in presentapplication.

All sequences, including reverse primers, are listed in their 5′ to 3′orientation. Start and stop positions on a reference HPV sequences aregiven in increasing value order. Hence:

-   -   concerning primers: start and stop positions are either the        start and stop positions of the reference HPV fragment, to which        the sequence of the primer corresponds (case of forward primer),        or the start and stop positions of the reference HPV target        fragment, to which the primer anneals (case of the reverse        primers);    -   with regards to the probes: as a probe and its complementary        sequence are, at least in simplex amplification, products, which        have equivalent functions, the start and stop positions are        either those of the reference HPV fragment, to which the        sequence of the probe corresponds, or those of the reference HPV        target fragment, to which the probe anneals.

DETAILED DESCRIPTION

The present invention is based on an approach, which is completelydifferent from, and truly innovative, compared to prior art techniques.The invention overcomes the drawbacks of prior art techniques, and hasnumerous advantages, notably in terms of clinical applicability,performance, reliance and flexibility.

As mentioned in “the above background” section, prior art primers aredesigned either as type-specific primers, or as general consensusprimers by classic alignment of as many mucosal HPV sequence asrequired, or desired, or as possible (e.g., by direct alignment of the13 HR HPV).

On the contrary, the primers of the invention have been designed by HPVgroups (=HPV genera).

Indeed, the present inventors analyzed the phylogeny of HPV, and haveconstructed the resulting phylogenic tree, which is shown in FIG. 1.

The present inventors selected one HPV sub-family, which is involved incarcinogenesis of the mucosal epithelia, namely sub-family A. Theyfurther selected a set of HPV types, which is representative of HPVsub-family A. This representative set consists in 35 different HPVtypes, among which 18 types are mucosal HPV, which are at leastpotentially oncogenic HPV (i.e., the 13 HPV known as the 13 HR; twopotentially HR HPV -HPV53 and HPV66-; and three other HPV, which arebelieved to have an oncogenic potential -HPV82, HPV67 and HPV85-), theremaining 17 other HPV types being up to now known as beingnon-oncogenic HPV (see FIG. 1).

The inventors thus came to the conclusion that those HPV, which have atropism for the mucosal epithelia, and which are at least potentiallyoncogenic HPV, are distributed among groups A6, A5, A9 and A7 (see FIG.1). More precisely, said mucosal oncogenic HPV are distributed among:

-   -   group A6, for HPV66, HPV56, HPV53;    -   group A5, for HPV51, HPV82;    -   group A9, for HPV58, HPV33, HPV67, HPV52, HPV35, HPV31, HPV16;    -   group A7, for HPV68, HPV39, HPV85, HPV59, HPV45, HPV18.

By “HPV, which is at least potentially oncogenic”, it is meant that saidHPV is either known to be oncogenic (such as those 13 HPV, which areusually referred to as the 13 HR HPV, as well as other oncogenic HPV,such as HPV66, HPV53, and HPV82), or which have been described at leastby some authors as potentially oncogenic (such as HPV85), or which wouldbe in the future described as associated to a tumorous mucosa.

The design by group is a special feature shared by all the products ofthe invention.

In addition to covering at least the five most common HR HPV types,preferably at least 7 HR HPV, still preferably the five most common HRHPV as well as at least two other HR HPV, advantageously at least twoother HR HPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31,16, 45, 18), more preferably at least the thirteen HR HPV types, in asingle-tube experiment, the means offered by the present invention islikely to detect any particular variant that a patient may have. Hence,the method of the invention is much safer than any prior art method.

In case of multiple HPV infections, several HPV types are present in thecollected sample. A competitive effect may then be observed, wherein oneHPV type takes prevalence over another one for the same consensusprimer. Detection of the competed HPV may then be hampered, although theprimer has initially been designed to hybridize to both HPV.

The present invention further has the advantage of enabling the analysisof multi- and/or co-infection cases, without any loss in specificity andsensitivity.

The present invention thus relates to amplification primer systems, todetection probes system, and to pharmaceutical compositions, biologicalcompositions, and detection kits comprising at least one of saidamplification and detection systems. The present invention also relatesto a method of HPV detection, which comprises the amplification of atleast one HPV nucleic acid fragment, by at least one amplificationprimer system of the invention, and which optionally comprises thedetection of the amplicon(s) thereby produced, by at least one detectionprobe system of the invention.

The HPV detection method of the invention is notably useful for thediagnosis of an HPV-related disease or condition, for the prognosis orrisk assessment of such an HPV-related disease or condition, formonitoring of the evolution of an HPV-related disease or condition, formonitoring the efficiency of an anti-HPV drug or treatment, such ase.g., an anti-HPV vaccine or an anti-HPV vaccine candidate (e.g., ananti-HPV16 and/or anti-HPV18 and/or anti-HPV45 vaccine, or vaccinecandidate).

Said HPV-related disease or condition is any disease or conditioninvolving HPV, and more particularly an HPV-related neoplasia (e.g.,cervical intraepithelial neoplasia) or cancer, such as a cervicalcancer.

The present invention thus provides amplification primer systems anddetection probe systems, which are targeted to the oncogenic HPV ofgroup A6, and/or to the oncogenic HPV of A5, and/or to the oncogenic HPVof A9, and/or to the oncogenic HPV of A7.

Most of the amplification and/or detection systems of the presentinvention are multiplex systems, which comprises more than two primersand/or more than one probe. It is notably the case of the A9-targetedsystems and of the A7-targeted systems.

Each amplification primer system can be implemented with anamplification primer system of another group in a single-tubeamplification assay, without any significant loss in specificity. Hence,at least one A6-targeted amplification primer system of the presentinvention and at least one A5-targeted amplification primer system ofthe present invention and at least one A9-targeted amplification primersystem of the present invention and at least one A7-targetedamplification primer system of the present invention, can be usedtogether in a single-tube amplification assay, without any significantloss in specificity.

For each amplification primer system, the invention provides at leastone detection probe system, thereby forming an amplification anddetection system (i.e., a real-time amplification system).

Each detection probe system of the present invention can be implementedwith its corresponding amplification primer system in a single-tubeassay, without any significant loss in specificity, thereby allowing fora real-time HPV amplification and detection.

The present invention thus provides with group-targeted amplificationand detection systems, namely:

-   -   several A6-targeted amplification and detection systems,    -   several A5-targeted amplification and detection systems,    -   several A9-targeted amplification and detection systems,    -   several A7-targeted amplification and detection systems.

Each amplification and detection system can be implemented with anamplification and detection system of another group in a single-tubeamplification assay, without any significant loss in specificity,thereby allowing for a single-tube multi-multiplex (or “megaplex”)real-time amplification and detection of those HPV, which have a tropismfor the mucosal epithelia, and which are at least potentially oncogenicHPV.

Hence, at least one A6-targeted amplification and detection system ofthe present invention, and at least one A5-targeted amplification anddetection system of the present invention, and at least one A9-targetedamplification and detection system of the present invention, and atleast one A7-targeted amplification and detection system of the presentinvention, can be used together in a single-tube amplification assay,without any significant loss in specificity.

Whilst the invention provides systems, which are especially adapted tomultiplex or multi-multiplex implementation, the implementation of asystem of the invention in simplex mode is of course also encompassed bythe present invention.

The amplification and detection systems of the present invention furtherhave levels of Ct and sensitivity, which are sufficiently homogeneous toallow for a quantitative HPV amplification and detection. The presentinvention thereby allows for the identification of the presence of oneor several mucosal HPV in a single-tube assay, as well as for thedetermination of the viral HPV load(s). The quantitative property of thepresent invention is retained, even when it is implemented in a“megaplex” mode.

The invention thus relates to group-targeted amplification and/ordetection systems, and to their use for the detection of mucosal HPV,said group-targeted amplification and/or detection systems sharing thespecial technical feature of being suitable for multiplex (in fact,multi-multiplex, i.e., “megaplex”) amplification and for real-timedetection thereof, whereby these systems allow for the detection in asingle-tube assay of at least the five most common HR HPV (i.e., HPV16,18, 45, 31, 33),

preferably at least 7 HR HPV,still preferably the five most common HR HPV as well as at least twoother HR HPV, advantageously at least two other HR HPV belonging togroups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18),even still preferably at least the thirteen HPV known as HR HPV (HPVtypes 56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59, 45 and 18),more preferably, at least the thirteen HPV as well as at least one amongHPV types 66, 82, 67, 85, and 53still more preferably, at least the thirteen HPV as well as at least twoamong HPV types 66, 82, 67, 85, and 53even still more preferably, at least the thirteen HPV as well as atleast three among HPV types 66, 82, 67, 85, and 53most preferably, at least the thirteen. HPV as well as at least fouramong HPV types 66, 82, 67, 85, and 53, notably of least the seventeenmucosal HPV, consisting of said 13 HR HPV and HPV types 66, 82, 67, 85,still most preferably, at least the thirteen HPV as well as at least thefive HPV types 66, 82, 67, 85, and 53.

The group-targeted amplification and/or detection systems of the presentinvention further share the special technical feature of allowing such areal-time “megaplex” amplification to be quantitative.

As above-mentioned, the amplification and detection systems of thepresent invention are based on a truly innovative group-based approach.

Indeed, according to prior art general consensus techniques, the designof the primers (also referred to as “universal” primers) is made byalignment of all HPV sequences to be amplified, e.g., the 13 HR HPV, anddetermination of consensus sequences, which targets as many of the 13HPV as possible. Hence, such consensus sequences are found in aconserved gene or region, such as a gene of late coding region (e.g.,gene L1), and the same conserved gene or region is selected for thewhole set of HPV to be amplified.

On the contrary, the present inventors made a design per HPV group,i.e., they selected appropriate targets for each HPV group. Moreparticularly, they selected to genes of the early coding region, moreparticularly genes E1, E2, E6, E7. The present inventors have designedparticular targets for each the desired HPV groups.

For example, the primers, which have been designed for group A5, havetheir target within genes E7, E1; the primers, which have been designedfor group A6, have their target within genes E6, E7; the primers, whichhave been designed for group A7, have their target within gene E1; andthe primers, which have been designed for group A9, have their targetwithin genes E1, E2. Illustrative targets of the invention are shown inFIGS. 2A (groups A5 and A6) and 2B (groups A7 and A9).

Hence, the present inventors selected reference template sequences foreach of groups A5, A6, A7 and A9, wherein the A5 reference templatesequences are within the region consisting of genes E7 and E1, the A6reference template sequences are within the region consisting of genesE6 and E7, the A7 reference template sequences are within gene E1, andthe A9 reference template sequences are within the region consisting ofgenes E1 and E2.

The primers are designed and built to hybridize to one end of saidtargets (or to the complementary sequence thereof), whereby a primerpair anneals to each end of said target (or to the complementarysequence thereof).

The probes are designed and built to anneal to one of said referencetemplate sequences, whereby each probe anneals to at least one of theamplicons generated by a primer system of the invention.

An amplification primer system of the invention comprises at least twoprimers. A detection probe system of the invention comprises at leastone probe.

The amplification primer systems of the invention preferentially amplifyHPV that belong to group A6 or A5 or A9 or A7, i.e., oncogenic HPV.

The present inventors further succeeded in producing amplificationprimer systems, which are specific of the HPV set formed by groups A5and A6 and A7 and A9. These amplification primer systems of theinvention do not amplify any HPV that would belong to a group other thanA5, A6, A7, A9. Indeed, most primer systems of the invention arespecific of the HPV set formed by groups A6 and A5 and A9 and A7.

Moreover, most of amplification primer systems of the invention arespecific of the group to which they are targeted, i.e., most primersystems of the invention are specific of group A6 or A5 or A9 or A7:most primers of a given amplification system amplify one or several HPVof the same HPV group, without amplifying any HPV that would belong toanother HPV group.

Those primer systems of the invention, which are not specific of the HPVset formed by groups A5 and A6 and A7 and A9, may amplify nucleic acidsthat are not from an HPV of said groups, e.g., a non-oncogenic HPV. Insuch a case, these primer systems of the invention do however show amuch lower amplification efficiency for these non-target amplicons(e.g., they lead to a PCR efficiency that is much lower than the oneobtained for HPV of groups A5 and/or A6 and/or A9 and/or A7, and aretherefore not quantitative).

When a group-targeted primer system of the invention is combined with aprobe system of the invention that is targeted to the same group, thegroup-targeted real-time amplification system that results therefrom isspecific of the HPV set formed by groups A5 and A6 and A9 and A7: noneof such real-time amplification systems detects an HPV that would notbelong to group A5 or A6 or A9 or A7. Furthermore, most of thesereal-time amplification systems are specific of the group to which theytargeted.

Only one real-time amplification system of the invention has across-group reactivity: the real-time amplification primer systemdesigned for group A9, which is referred to in the examples below assystem C, detects the seven HPV types of group A9, and also HPV53, whichis an oncogenic HPV belonging to group A6 (HPV53 being a potentially HRHPV); this group A9 system C does however not detect any other HPV amongthe 42 HPV tested (no group A6 HPV other than HPV53; no group A5 HPV; nogroup A11 HPV; no group A7 HPV, no group A4 HPV; no group A3 HPV).

In any case, does a real-time amplification system of the presentinvention detect a nucleic acid that would be a human nucleic acid.

When it is desired to amplify any HPV, which belongs to group A6, A5,A9, or A7, most of the amplification primer systems of the presentinvention will in fact comprise more than two primers, i.e., at leastthree primers.

For example, the amplification primer systems, which are targeted togroup A6 or A5, only require a primer pair to amplify HPV56 andoptionally HPV66 (for group A6), or HPV51 and optionally HPV82 (forgroup A5). But, those amplification primer systems, which are targetedto group A9 or A7, have to amplify six or seven HPV types of group A9,or five or six HPV types of group A7, if a complete coverage of the HPVspectrum of the group is desired. Under such instances, an A9- orA7-targeted amplification system of the invention may comprise e.g., atleast three or four forward primers, and two, three, four, five or morereverse primers. Such A9- or A7-targeted amplification primer systemstherefore are already multiplex systems by themselves.

Of course, the skilled person, who would like to restrict the spectrumof amplified HPV, might select fewer forward and/or reverse primers,depending on which HPV types or strain he/she would like to amplify.

Similarly, when it is desired to detect any HPV, which belongs to groupA6, A5, A9, or A7, most of the detection probe systems of the presentinvention will in fact comprise more than one probe, i.e. at least twoprobes.

For example, the detection probe systems, which are targeted to group A6or A5, only require probe to detect HPV56 and optionally HPV66 (forgroup A6), or HPV51 and optionally HPV82 (for group A5). But, thosedetection probe systems, which are targeted to group A9 or A7, maycomprise one probe per HPV type or strain to be detected, notably when aboth a complete coverage of the HPV group spectrum, and a simultaneousHPV group or type discrimination are desired. An A9- or A7-targeteddetection probe system may thus comprise at least four probes, forexample four, five or six probes.

As above-mentioned, all detection probe systems of the present inventionare adapted to real-time detection, and can thus be implemented withtheir corresponding amplification primer systems in the very same tube.

In the examples below, are described several A6-, A5-, A9 andA7-targeted amplification and/or detection systems of the invention.

In these examples, are shown table 12 to table 88:

-   -   tables 12-35: these tables gives the SEQ ID NO: and positions of        the reference amplicons (the sequences of these reference        template amplicons are also listed after the last table, i.e.,        before the “claims” section), the forward primers, the reverse        primers, the probes, the beacons probes of illustrative        group-targeted systems of the invention;    -   tables 35-50: these tables show the number of nucleotide        mismatches shown by primers and probes of the invention        (alignment of the sequences of 50 HPV types); an empty box        indicates there is no coherent sequence alignment;    -   tables 51-68: specificity of the detection systems of the        invention;    -   tables 69-82: system sensitivity;    -   tables 83-88: “megaplex” specificity and sensitivity,    -   table 89: list of HPV genome sequences.

A reference genome has been elected for each HPV group, namely:

-   -   for group A5: the reference genome is the genome sequence of        HPV51 available under accession number NC_(—)001533.1,    -   for group A6: the reference genome is the genome sequence of        HPV56 available under accession number NC_(—)001594.1,    -   for group A7: the reference genome is the genome sequence of        HPV18 available under accession number NC_(—)001357.1,    -   for group A9: the reference genome is the genome sequence of        HPV16 available under accession number NC_(—)001526.1,

In the following tables, the term <<address>> means a nucleotideposition in the reference genome. More precisely:

-   -   a forward primer having an address X is an isolated        oligonucleotide, which has a sequence which is the sequence of a        fragment of the reference genome starting at position X in said        reference genome, or a variant of such a fragment sequence (see        the mismatch count tables below),    -   a reverse primer having an address X is an isolated        oligonucleotide, which has a sequence which is complementary to        the sequence of a fragment of the reference genome ending at        position X in said reference genome, or a variant of such a        fragment sequence (see the mismatch count tables below),    -   a probe having an address X is an isolated oligonucleotide, the        sequence of which is the sequence of a fragment of the reference        genome starting at position X in said reference genome, or which        is the complementary sequence of such a fragment sequence (over        the entire length of this fragment sequence), or a variant of        such a fragment sequence or of such a complementary fragment        sequence (see the mismatch count tables below).

As a consequence, when a primer pair, which is selected from a givensystem, to consist of a forward primer having the address Xf, and of areverse primer having the address Xr, is implemented on its referenceHPV genome under conditions favorable to nucleic acid amplification,this primer pair then amplifies from said HPV reference genome anamplicon, which consists of the sequence that extends from position Xfto position Xr in said reference HPV genome (start and stop positionsincluded).

When this primer pair is, under conditions favorable to nucleic acidamplification, implemented on a given HPV genome, which is not itsreference HPV genome, but which belongs to the same group as thisreference HPV genome, this primer pair then amplifies from said givenHPV genome an amplicon, which consists in the sequence of the fragmentof said given HPV genome, which corresponds by sequence alignment to thefragment that extends from position Xf to position Xr in said referenceHPV genome.

A fragment. A, which is a fragment of a given HPV genome and, whichcorresponds by sequence alignment to a fragment B of a HPV referencegenome, means that said fragment A has the same length as said fragmentB, and that the start and stop positions of said fragment A within saidgiven HPV genome are such that, among those fragments of said given HPVgenome, which have the same length as said fragment B, said fragment Ais the fragment, which gives the best identity score when compared tothe nucleotide sequence of said fragment B, as determined by globalalignment to the nucleotide sequence of fragment B.

It will also be understood that:

-   -   a forward primer having an address Xf and a length Lf is an        isolated oligonucleotide, which has a sequence which is the        sequence of a fragment of the reference genome starting at        position Xf in said reference genome, and ending at position        Xf+Lf−1 in said reference genome, or a variant of such a        fragment sequence (see the alignments shown below),    -   a reverse primer having an address Xr and a length Lr is an        isolated oligonucleotide, which has a sequence which is        complementary to the sequence of a fragment of the reference        genome starting at position Xr−Lr−1, and ending at position Xr        in said reference genome, or a variant of such a fragment        sequence (see the alignments shown below),    -   a probe having an address Xp and a length Lp is an isolated        oligonucleotide, the sequence of which is the sequence of a        fragment of the reference genome starting at position Xp and        ending at position Xp+Lp−1 in said reference genome, or which is        complementary to such a fragment sequence, or a variant of such        a fragment sequence or of such a complementary fragment sequence        (see the alignments shown below).

The present invention thus relates to a process for the nucleic acidamplification of at least one HPV target, wherein said HPV is an HPV,which has a tropism for the mucosal epithelia, and which is at leastpotentially oncogenic, preferably at least one oncogenic anogenital HPVtarget, most preferably at least one oncogenic cervical HPV. Theamplification process of the invention comprises the step of producingfrom said at least one HPV target at least one amplicon by means of atleast two primers.

According to an advantageous embodiment of the present invention, theamplification process of the present invention can be a real-timemultiplex amplification process, involving at least one probe that cananneal to the amplicon(s) generated by said t least two primers.

The present invention relates to a process for the detection of at leastone HPV, which has a tropism for the mucosal epithelia, and which is anat least potentially oncogenic HPV, preferably at least one oncogenicanogenital HPV, most preferably at least one oncogenic cervical HPV, ina sample. The detection process of the present invention comprises thedetection of at least one nucleic acid HPV target which has beenamplified by the process of nucleic acid amplification of the invention.

The present invention relates to a process for the detection of at leastone HPV, which has a tropism for the mucosal epithelia, and which is anat least potentially oncogenic HPV, preferably at least one oncogenicanogenital HPV, most preferably at least one oncogenic cervical HPV, ina sample, by determination of whether at least one amplicon has been, oris produced from said sample, or from nucleic acid material thereof, byamplification by means of at least two amplification primers of theinvention, and/or at least one amplification primer system of theinvention.

The production of at least one amplicon indicates that at least one HPV,which has a tropism for the mucosal epithelia, and which is at leastpotentially oncogenic, preferably at least one oncogenic anogenital HPV,most preferably at least one oncogenic cervical HPV, is present in saidsample.

According to an advantageous embodiment of the present invention, thedetermination of whether at least one amplicon is produced can becarried out in real-time multiplex amplification, preferably with atleast one probe of the invention and/or at least one probe system of theinvention.

The present invention thus relates to a process for the detection of atleast one HPV, which has a tropism for the mucosal epithelia, and whichis at least potentially oncogenic, preferably at least one oncogenicanogenital HPV, most preferably at least one oncogenic cervical HPV, ina sample, which comprises:

-   -   contacting said sample, or nucleic acid material thereof, with        at least two amplification primers of the invention and/or at        least one primer system of the invention, under conditions        suitable to the production of at least one amplicon by said        primers (i.e., under conditions, which would be suitable to the        production by said primers of at least one amplicon from said at        least one HPV to be detected, if this HPV were present in said        sample),    -   determining whether at least one amplicon has been produced, or        is produced by said primers (e.g., by means of at least one        detection probe, preferably in real-time amplification involving        at least one probe of the invention and/or at least one        detection probe system of the invention).

The production of at least one amplicon indicates that at least one HPV,which has a tropism for the mucosal epithelia, and which is an at leastpotentially oncogenic HPV, preferably at least one oncogenic anogenitalHPV, most preferably at least one oncogenic cervical HPV, is present insaid sample.

By “sample containing nucleic acid material”, it is meant any sample,which contains at least one nucleic acid, e.g., a biological sample,such as a sample which has been collected from a cell culture, or froman animal or a human being, e.g., from a female human being, preferablya sample which has been collected from a uterine cervix.

Said sample may optionally have been further treated and/or purifiedaccording to any technique known by the skilled person, to improve theamplification efficiency and/or qualitative accuracy and/or quantitativeaccuracy. The sample may thus exclusively, or essentially, consist ofnucleic acid(s), whether obtained by purification, isolation, or bychemical synthesis. Means are available to the skilled person, who wouldlike to isolate or purify nucleic acids, such as DNA, from a biologicalsample, for example to isolate or purify DNA from cervical scrapes(e.g., QIAamp-DNA Mini-Kit; Qiagen, Hilden, Germany).

Hence, the detection method of the present invention enables thereal-time multiplex detection, preferably the real-time quantitativemultiplex detection of:

at least the five most common HR HPV (i.e., HPV16, 18, 45, 31, 33),preferably at least 7 HR HPV,still preferably the five most common HR HPV as well as at least twoother HR HPV, advantageously at least two other HR HPV belonging togroups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18),even still preferably at least the thirteen HPV known as HR HPV (HPVtypes 56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59, 45 and 18),more preferably, at least the thirteen HPV as well as at least one amongHPV types 66, 82, 67, 85, and 53still more preferably, at least the thirteen HPV as well as at least twoamong HPV types 66, 82, 67, 85, and 53even still more preferably, at least the thirteen HPV as well as atleast three among HPV types 66, 82, 67, 85, and 53most preferably, at least the thirteen HPV as well as at least fouramong HPV types 66, 82, 67, 85, and 53, notably of least the seventeenmucosal HPV, consisting of said 13 HR HPV and HPV types 66, 82, 67, 85,still most preferably, at least the thirteen HPV as well as at least thefive HPV types 66, 82, 67, 85, and 53,in a single-tube assay.

The present invention also relates to all the medical, biological,pharmaceutical applications of the detection method of the invention,and/or of the primers and/or probes of the invention.

The present invention thus relates to a process for the diagnosis orprognosis of cervical neoplasia or cancer, which comprises determiningthe presence, re-occurrence, persistence, or cellular spread of at leastone HPV by the detection method of the invention, e.g., by implementingit on a sample, which may have been collected from a patient, whereby apositive determination indicates that there is a cervical neoplasia orcancer, or that there is a prevalent risk of such a condition ordisease.

The present invention also relates to a process for monitoring theefficiency of an anti-HPV treatment or drug, or an anti-HPV candidatetreatment or drug, such as an anti-HPV16 and/or 18 and/or 45 treatment,drug, candidate treatment or candidate drug, which comprises determiningsaid treatment, drug, candidate treatment or candidate drug induces thenon-reoccurrence, non-persistence, disappearance, or a decrease incellular spread of at least one HPV by the detection method of theinvention, whereby a positive determination indicates that saidtreatment, drug, candidate treatment or candidate drug is an efficientanti-HPV drug.

The present invention also relates to a method to produce an anti-HPVdrug, which comprises:

-   -   providing at least one anti-HPV candidate drug,    -   administering said at least one candidate anti-HPV drug to a        cell culture or to a non-human animal, wherein said cell culture        or animal is or comprises at least one cervical neoplasia or        cancer, and    -   determining by the HPV detection method of the invention whether        said candidate anti-HPV drug induces the regression or        disappearance of said at least one neoplasia or cancer,        whereby a positive determination indicates that said candidate        drug is an efficient anti-HPV drug.

As above-mentioned, the present invention thus provides HPVamplification and detection means, which can be implemented in real-timeand in multiplex, i.e., which combine target amplification and detectionin one single operative step, and which enables the detection of mucosalHPV in one single operative step, without any significant loss inspecificity.

Furthermore, the amplification and detection systems of the inventionhave Ct and sensitivity levels, which are sufficiently homogeneous toallow for a real-time quantitative multiplex HPV detection.

According to the present invention, said amplification primers maycomprise:

-   -   at least two primers, which are intended for targeting oncogenic        HPV of group A6, wherein said at least two A6-targeted primers        are oligonucleotides, which consist of 14-30 nucleotides,        preferably of 15-29, more preferably of 16-28, most preferably        of 17-25 nucleotides, the sequences of which are suitable for        use as forward and reverse primers, respectively, in the        amplification of at least one nucleic acid of 90-390        nucleotides, preferably of 95-385 nucleotides, more preferably        of 100-379 nucleotides, from the A6-target region consisting of        the E6 and E7 genes of HPV56, and/or    -   at least two primers, which are intended for targeting oncogenic        HPV of group A5, wherein said at least two A5-targeted primers        are oligonucleotides, which consist of 14-30 nucleotides,        preferably of 15-29, more preferably of 16-28, most preferably        of 17-25 nucleotides, the sequences of which are suitable for        use as forward and reverse primers, respectively, in the        amplification of at least one nucleic acid of at least 90-240        nucleotides, preferably of 100-230 nucleotides, more preferably        of 106-225 nucleotides, from the A5-target region consisting of        the E7 and E1 genes of HPV51, and/or    -   at least two primers, which are intended for targeting oncogenic        HPV of group A9, wherein said at least two A9-targeted primers        are oligonucleotides, which consist of 14-30 nucleotides,        preferably of 15-29, more preferably of 16-28, most preferably        of 17-25 nucleotides, the sequences of which are suitable for        use as forward and reverse primers, respectively, in the        amplification of at least one nucleic acid of at least 80-260        nucleotides, preferably of 85-250 nucleotides, more preferably        of 88-241 nucleotides, from the A9-target region consisting of        the E1 and E2 genes of each of the following group A9 HPV:        HPV58, HPV33, HPV52, HPV35, HPV31 and HPV16, preferably from the        E2 gene of each of said group A9 HPV, and/or    -   at least two primers, which are intended for targeting oncogenic        HPV of group A7, wherein said at least two A7-targeted primers        are oligonucleotides, which consist of 14-30 nucleotides,        preferably of 15-29, more preferably of 16-28, most preferably        of 17-25 nucleotides, the sequences of which are suitable for        use as forward and reverse primers, respectively, in the        amplification of at least one nucleic acid of at least 100-220        nucleotides, preferably of 120-215 nucleotides, more preferably        of 125-209 nucleotides, from the A7-target region consisting of        the E1 gene of each of the following group A7 HPV: HPV68, HPV39,        HPV59, HPV45, and HPV18.

The nucleic acid which is amplified from said A6 or A5 or A9 or A7target region corresponds in said HPV to the nucleic acid sequence ofthe A6 or A5 or A9 or A7 reference template, respectively. Hence, theseamplified nucleic acids usually have a high degree of identity withtheir respective reference template sequences, e.g., an identity of atleast 80%, preferably of at least 85%, more preferably of at least 90%,most preferably of at least 95%, over the entire length of saidrespective reference template sequence.

By nucleic acid, it herein preferably meant DNA.

Preferably, the at least two primers, which are intended for targetingone HPV group, are different from the at least two primers, which areintended for targeting the three other groups.

Said at least two A6-targeted primers may target a sequence, which isentirely within the E6 gene of each of said group A6 HPV (namely,HPV56), or which is entirely within the E7 gene of each of said group A6HPV, or which overlap the E6 and E7 genes, for example with a forwardprimer targeting E6 and the reverse primer targeting E7, or conversely.Preferably, said at least two A6-targeted primers target a sequence,which overlap the E6 and E7 genes, for example with a forward primertargeting E6 and the reverse primer targeting E7, or conversely.

Said at least two A5-targeted primers may target a sequence, which isentirely within the E7 gene of each of said group A5 HPV (namely,HPV51), or which is entirely within the E1 gene of each of said group A5HPV, or which overlap the E7 and E1 genes, for example with a forwardprimer targeting E7 and the reverse primer targeting E1, or conversely.

Said at least two A9-targeted primers may target a sequence, which isentirely within the E1 gene of each of said group A9 HPV (namely, of atleast each of HPV58, HPV33, HPV52, HPV35, HPV31 and HPV16), or which isentirely within the E2 gene of each of said group A9 HPV, or whichoverlap the E1 and E2 genes, for example with a forward primer targetingE1 and the reverse primer targeting E2, or conversely. In the examplesgiven below, all A9 amplification systems target the E2 gene, exceptsystem C, which has a target that overlaps the E1 and E2 genes.

Said at least two A7-targeted primers may target a sequence, which isentirely within the E1 gene of each of said group A7 HPV (namely, withinthe E1 gene of at least each of HPV68, HPV39, HPV59, HPV45, HPV18).

In accordance with the present invention, said at least two A6-, A5-,A9- and A7-targeted primers notably share the specific technical featureof being suitable for use in a real-time (quantitative) multiplexdetection of HPV, which can be oncogenic for the mucosal epithelia.

By “consisting of 14-30 nucleotides”, it is meant “consisting of 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides”.

The same applies mutatis mutandis to any range, which is recited in thepresent application.

The nucleotide lengths of the primers can be chosen independently fromeach other.

By oligonucleotide or primer, “the sequence of which is suitable for usein the amplification of” at least one HPV, or nucleic acid or sequence,it is meant that the sequence of the oligonucleotide or primer is suchthat they can hybridize to this HPV or nucleic acid or sequence, underconditions of moderate, but preferably high or very high stringency.

A primer of the invention may consist of a 1430 nt oligonucleotide(preferably a 17-25 nt oligonucleotide), the sequence of which has anidentity of at least 80%, preferably of at least 85%, more preferably ofat least 90%, most preferably of at least 92%, with a sequence of thesame length contained in its group reference template sequence, mostpreferably with a sequence of the same length contained at the very 3′end or at the very 5′ end of said group reference template sequence.

According to an advantageous embodiment of the present invention, saiddetermination of whether at least one amplicon is produced can becarried out by using in real-time amplification at least one probe,preferably at least one A6-targeted probe and/or at least one at leastone A5-targeted probe and/or at least one A9-targeted probe and/or atleast one A7-targeted probe.

Preferably, said at least two A6-targeted primers are suitable for usein the amplification of more than one oncogenic HPV of group A6, namelyat least HPV56 and at least one other oncogenic HPV of group A6 (e.g.,HPV66, HPV53). According to the present invention, said at least twoA6-targeted primers anneal to each of the group A6 HPV they target, intheir E6 and/or E7 genes. Hence, according to an advantageous embodimentof the present invention, said at least two A6-targeted primers areoligonucleotides, the respective sequences of which are suitable for useas forward and reverse primers in the amplification of at least onenucleic acid from the region consisting of the E6 and E7 genes of eachof the following group A6 HPV: HPV56 and HPV66. HPV66 is at present timenot listed within the 13 HR HPV; some authors however consider thatHPV66 could be a HR HPV.

Preferably, said at least two A5-targeted primers are suitable for usein the amplification of more than one oncogenic HPV of group A5, namelyat least HPV51 and at least one other oncogenic HPV of group A5 (e.g.,HPV82). According to the present invention, said at least twoA5-targeted primers anneal to each of the group A5 HPV they target, intheir E7 and/or E1 genes. Hence, according to an advantageous embodimentof the present invention, said at least two A5-targeted primers areoligonucleotides, the respective sequences of which are suitable for useas forward and reverse primers in the amplification of at least onenucleic acid from the region consisting of the E7 and E1 genes of eachof the following group A5 HPV: HPV51 and HPV82.

Preferably, said at least two A9-targeted primers are suitable for usein the amplification of more than the six above-mentioned oncogenic HPVof group A9, namely at least HPV58, HPV33, HPV52, HPV35, HPV31 andHPV16, and at least one other oncogenic HPV of group A9 (e.g., HPV67).According to the present invention, said at least two A9-targetedprimers anneal to each of the group A9 HPV they target, in their E1and/or E2 genes. Hence, according to an advantageous embodiment of thepresent invention, said at least two A9-targeted primers areoligonucleotides, the respective sequences of which are suitable for useas forward and reverse primers in the amplification of at least onenucleic acid from the region consisting of the E1 and the E2 genes ofeach of the following group A9 HPV: HPV58, HPV33, HPV67, HPV52, HPV35,HPV31 and HPV16.

A group-targeted primer of the present invention may further target aHPV, which does not belong to the same group (as long as it does nottarget any group other than A6, A5, A7 or A9), i.e., an A9-targetedprimer pair may target an HPV belonging to group A6 (such as HPV53), inaddition to targeting the above-mentioned HPV of group A9. Hence, therespective sequences of said at least two A9-targeted primers mayfurther be suitable for use as forward and reverse primers in theamplification of at least one nucleic acid of HPV53.

Preferably, said at least two A7-targeted primers are suitable for usein the amplification of more than the five above-mentioned oncogenic HPVof group A7, namely at least HPV68, HPV39, HPV59, HPV45, and HPV18, andat least one other oncogenic HPV of group A7 (e.g., HPV85). According tothe present invention, said at least two A7-targeted primers anneal toeach of the group A7 HPV they target, in their E1 gene. Hence, accordingto an advantageous embodiment of the present invention, said at leasttwo A7-targeted primers are oligonucleotides, the respective sequencesof which are suitable for use as forward and reverse primers in theamplification of at least one nucleic acid from the E1 gene of each ofthe following group A7 HPV: HPV68, HPV39, HPV85, HPV59, HPV45, andHPV18.

To the best of the inventors' knowledge, there is no prior art multiplexprocess, which would allow the detection of a mucosal oncogenic HPVother than HPV56, HPV51, HPV58, HPV33, HPV52, HPV35, HPV31 and HPV16,HPV68, HPV39, HPV59, HPV45, and HPV18. Until recently, these thirteenHPV were considered to be the 13 HR HPV (i.e., the 13 high risk HPV,i.e., the HPV which have the highest tumor prevalence). However, otherHPV are now thought to belong to the HR HPV group, e.g., HPV66 and HPV53(group A6). As prior art techniques, such as the HPV Amplicor test, arebased on a global consensus design, additional HPV cannot beencompassed. On the contrary, the present invention is based on a designby group, and cover more than the 13 basic HR HPV, which is much saferfor the patient, and which has the special advantage of being evolutive,in the sense that any oncogenic A6, A5, A9 and A7 new corner are likelyto be taken into account.

Hence, to the best of the inventors' knowledge, the present inventionhas a mucosal oncogenic HPV spectrum, which is broadest than any otherprior art technique, and has a bigger potential of adaptability to anyevolution of HPV spectrum to be detected. The present inventiontherefore is safer than any other prior art technique.

The amplification primer systems of the invention preferentially amplifyHPV that belong to group A6 or A5 or A9 or A7, i.e., oncogenic HPV.

As above-mentioned, most of amplification primer systems of theinvention are specific of the HPV set formed by groups A5 and A6 and A7and A9, and more particularly are specific of the group to which theyare targeted, i.e., most primer systems of the invention are specific ofgroup A6 or A5 or A9 or A7 (and do not amplify any HPV of groups A11 orA4 or A3).

Hence, said at least two primers can advantageously be specific of theHPV set formed by groups A5 and A6 and A7 and A9, and more particularlyof group A6, or group A5, or group A9, or group A7, preferably of theoncogenic HPV of group A6, or group A5, or group A9, or group A7.

Hence, according to a preferred embodiment of the present invention,said at least two primers can have such sequences that they are notsuitable for use as forward and reverse primers in the amplification ofany nucleic acid from an HPV, which would not belong to group A6, A5, A9or A7. According to a more preferred embodiment of the presentinvention, the respective sequences of said at least two primerstherefore are not suitable for use as forward and reverse primers in theamplification of a nucleic acid from an HPV, which is not oncogenic,preferably which is not oncogenic for the mucosal epithelia, morepreferably which is not an anogenital HPV, most preferably which is nota cervical HPV.

In other words, said at least two primers preferably are specific ofoncogenic HPV, compared to non-oncogenic HPV.

The present invention can be implemented in simplex, multi-tube simplex,in multiplex, as well as in multi-multiplex (“megaplex”). According toan advantageous embodiment of the present invention, said amplificationcan be a single-tube multiplex amplification, or a single-tubemulti-multiplex amplification (“megaplex”).

By “single-tube multiplex amplification” or “multiplex amplification”,it is herein meant any amplification reaction aiming at theamplification, and optionally the detection, of more than one target inthe same tube. For instance, multiplex amplification include duplexamplification (two targets), triplex amplification (three targets), aswell as higher multiplex amplification. Multiplex amplification includesamplification reactions with more than one primer pair, for instance twoprimer pairs. In this case, there might be four different primers, butit is also possible for the two primer pairs to have one primer incommon, e.g., the forward primer, and to have two distinct reverseprimers. Multiplex amplification and detection also includesamplification reactions with a unique primer pair, but with more thanone probe.

Hence, according to the multiplex embodiment of the present invention,more than one primer pair is present in the amplification reactionmixture. As a very advantageous embodiment of the present invention, atleast four, preferably at least six, more preferably at least eightprimer pairs can be present in the amplification reaction mixture.Indeed, the primers of the invention allow for a multiplexamplification, without any significant specificity loss. Hence, allreagents can be placed in the same tube to carry out the amplificationassay on the sample to be tested, whereby whatever mucosal oncogenic HPVis present in this sample, it will be detected in a single-stepprocedure.

Preferably, said amplification primers comprise said at least twoA6-targeted primers, and at least two primers selected from:

-   -   said at least two A5-targeted primers,    -   said at least two A9-targeted primers, and    -   said at least two A7-targeted primers.

Preferably, said amplification primers comprise said at least twoA5-targeted primers, and at least two primers selected from:

-   -   said at least two A6-targeted primers, and/or    -   said at least two A9-targeted primers, and/or    -   said at least two A7-targeted primers.

Preferably, said amplification primers comprise said at least twoA9-targeted primers, and at least two primers selected from:

-   -   said at least two A5-targeted primers, and/or    -   said at least two A6-targeted primers, and/or    -   said at least two A7-targeted primers.

Preferably, said amplification primers comprise said at least twoA7-targeted primers, and at least two primers selected from:

-   -   said at least two A5-targeted primers, and/or    -   said at least two A9-targeted primers, and/or    -   said at least two A6-targeted primers.

Most preferably, said amplification primers comprise:

-   -   said at least two A6-targeted primers, and    -   said at least two A5-targeted primers, and    -   said at least two A9-targeted primers, and    -   said at least two A7-targeted primers.

According to an advantageous embodiment of the present invention, saiddetermination of whether at least one amplicon is produced, is carriedout by means of at least one probe, which is intended to anneal to saidat least one amplicon, i.e., the sequence of the probe is sufficientlycomplementary to said at least one amplicon (or to the complementarysequence thereof) that the probe can anneal to said at least oneamplicon, preferably under conditions of moderate, high or very highstringency.

The probe(s) of the invention can be implemented in any appropriateformat. Preferably, the probe(s) of the invention is(are) notimmobilized onto a solid support.

Most preferably, the probe(s) of the invention is(are) used in real-timeamplification.

Hence, according to an advantageous embodiment of the present invention,said amplification can be a real-time amplification.

According to an advantageous embodiment of the present invention, saidat least one probe is used in real-time amplification, i.e., said atleast two primers and said at least two probes can be both present inthe amplification reaction mixture, whereby said at least one probeanneal to the amplicon(s) produced by said at least two primers inreal-time. In other words, the amplification and the detection can becarried out in a single step, namely in real-time amplification.

By real-time amplification, we hereby understand any amplification-basedprocess allowing for monitoring of fluorescence emitted during thereaction as an indicator of amplicon production during eachamplification cycle as opposed to the endpoint detection by conventionalamplification process.

The present invention provides probes, which are intended to target theamplicon(s) obtainable by amplification of a mucosal oncogenic HPVnucleic acid by at least two A6- and/or A5- and/or A9- and/orA7-targeted primers.

These probes share the specific technical features of being suitable foruse in a real-time multiplex amplification.

Some probes of the present invention allow to detect all the HPV of onegroup (i.e., they anneal to every amplicon that is obtainable by meansof a group-targeted primer pair of the invention). Other probes of thepresent invention detect one or only some HPV of one group. Hence, thepresent invention further provides different levels of detection: theresponse can be a global detection of the presence or absence of atleast one mucosal oncogenic HPV, or a more precise response, such aspresence or absence of at least one mucosal oncogenic HPV of group A6and/or A5 and/or A9 and/or A7, or an even more precise response such aspresence or absence of at least one mucosal oncogenic HPV type(s).

Preferably, said determination of whether at least one amplicon isproduced, is carried out by using in real-time amplification at leastone A6- and/or A5- and/or A9- and/or A7-targeted probe, the sequence ofwhich is suitable for use as a probe for the detection of at least oneamplicon produced by said at least two A6- and/or A5- and/or A9- and/orA7-targeted primers, respectively.

According to an advantageous embodiment of the present invention, saidamplification can be a real-time multiplex amplification.

According to a very advantageous embodiment of the present invention,said amplification can be carried out as a real-time multiplexamplification. Hence, the amplification reaction mixture can comprisemore than two primers, and also at least one probe, without anysignificant loss of specificity in the detection of HPV. To the best ofthe inventors' knowledge, the present technique is the first to allow areal-time multiplex amplification. It is very advantageous, in the sensethat any mucosal oncogenic HPV can be screened in a single tube assay,wherein all reactants (primers and probe(s)) have been poured. Moreparticularly, the present invention provides A6-, A5, A9- andA7-targeted primers and probes, which can all be placed in the samereaction mixture, without any significant loss in specificity.

The present invention therefore is much easier-to-handle and muchquicker than any prior art technique. It further limits the possibilityof any experimental error or contamination in the sample analysis.

To the best of the inventors' knowledge, the present technique is thefirst to enable a real-time multiplex amplification, which allows tocover at least the five most common HR HP V (HPV16, 18, 45, 31, 33),preferably at least 7 HR HPV, still preferably the five most common HRHPV as well as at least two other HR HPV, advantageously at least twoother HR HPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31,16, 45, 18), more preferably at least the 13 HR HPV, and asabove-mentioned, most preferably at least 18 oncogenic HPV (namely, atleast the 13 HR and five other oncogenic HPV, i.e., HPV 66, 53, 82, 67,85).

According to an advantageous embodiment of the present invention, saidamplification can be a quantitative real-time multiplex amplification.

According to an even more advantageous embodiment of the presentinvention, said amplification can be carried out as a quantitativereal-time multiplex amplification. Indeed, the primers and probes of thepresent invention have such a sequence that there is no loss inspecificity and no loss in quantitative accuracy, even when they areimplemented in real-time multiplex amplification.

To the best of the inventors' knowledge, the present technique is thefirst to enable a quantitative real-time multiplex amplification, whichallows to cover at least the five most common HR HPV (HPV16, 18, 45, 31,33), preferably at least 7 HR HPV, still preferably the five most commonHR HPV as well as at least two other HR HPV, advantageously at least twoother HR HPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31,16, 45, 18), more preferably at least the 13 HR HPV, and asabove-mentioned, most preferably at least 18 oncogenic HPV (namely, atleast the 13 HR and five other oncogenic HPV, i.e., HPV 66, 53, 82, 67,85).

The invention thereby finds applications not only in the field ofdiagnostic, but also in the field of therapy evaluation, such as tomonitor the efficiency of an anti-HPV treatment, or to evaluate theefficiency of an anti-HPV drug. To the best of the inventors' knowledge,such therapy-related applications were previously unattainable by any ofthe prior art process.

The present invention thus represents a technological breakthrough inthe field of HPV monitoring.

Hence, said amplification can be a quantitative real-time multiplexamplification, which allows for the detection of one or several of HPV,which can be oncogenic for the mucosal epithelia, in a single-tubeamplification run.

The present invention thereby allows for the real-time multiplexdetection, preferably the real-time quantitative multiplex detection of:

at least the five most common HPV (HPV16, 18, 45, 31, 33)preferably at least 7 HR HPV,still preferably the five most common HR HPV as well as at least twoother HR HPV, advantageously at least two other HR HPV belonging togroups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18),even still preferably at least the thirteen HPV known as HR HPV (HPVtypes 56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59, 45 and 18),more preferably, at least the thirteen HPV as well as at least one amongHPV types 66, 82, 67, 85, and 53still more preferably, at least the thirteen HPV as well as at least twoamong HPV types 66, 82, 67, 85, and 53even still more preferably, at least the thirteen HPV as well as atleast three among HPV types 66, 82, 67, 85, and 53most preferably, at least the thirteen HPV as well as at least fouramong HPV types 66, 82, 67, 85, and 53, notably of least the seventeenmucosal HPV, consisting of said 13 HR HPV and HPV types 66, 82, 67, 85,still most preferably, at least the thirteen HPV as well as at least thefive HPV types 66, 82, 67, 85, and 53,in a single-tube assay.

Preferably, the respective sequences of said at least two A6-targetedprimers are suitable for use in the amplification of at least onereference template sequence, wherein said at least one referencetemplate sequence is a fragment consisting of positions 413-791 (SEQ IDNO:337) of the HPV56 sequence of SEQ ID NO:420 (accessionNC_(—)001594.1); or a conservative sub-fragment thereof, which hasretained the property of being a suitable reference template sequence,to construct and produce A6-targeted primers, which allow for areal-time multiplex detection of those HPV, which can be oncogenic forthe mucosal epithelia, preferably of at least the five most common HPV(HPV16, 18, 45, 31, 33), still preferably at least 7 HR HPV, even stillpreferably the five most common HR HPV as well as at least two other HRHPV, advantageously at least two other HR HPV belonging to groups A6and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), more preferably atleast the 13 HR HPV, most preferably at least the 13 HR HPV and at leastone, at least two, at least three, at least four, or the five of HPV66,53, 82, 67, 85; or a sequence which is fully complementary to saidfragment or sub-fragment over the entire length of said fragment orsub-fragment.

More preferably, the respective sequences of said at least twoA6-targeted primers are suitable for use in the specific amplificationof at least one reference template sequence, which consists of one ofSEQ ID NO:25-29 and NO:334-338, as shown in Table 18 (A6 referencetemplates); or a sequence which is fully complementary thereto over theentire length of said SEQ ID NO sequence.

Said reference template sequences notably share the specific technicalfeature of being suitable references to construct and produceA6-targeted primers, which allow for a real-time multiplex detection ofHPV, which can be oncogenic for the mucosal epithelia, preferably of atleast the five most common HPV (HPV16, 18, 45, 31, 33), still preferablyat least 7 HR HPV, even still preferably the five most common HR HPV aswell as at least two other HR HPV, advantageously at least two other HRHPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45,18), more preferably at least the 13 HR HPV, most preferably at leastthe 13 HR HPV and at least one, at least two, at least three, at leastfour, or the five of HPV66, 53, 82, 67, 85, and preferably for areal-time quantitative multiplex detection of such HPV.

As the reference template sequence is defined as consisting of one ofthe above-mentioned SEQ ID NOs (and not as comprising one of theabove-mentioned SEQ ID NOs), the primers do not flank these referencetemplate sequences, but fall within the reference template sequence, insuch a way that the amplicon consists of one of the listed SEQ ID NOs.Unless otherwise stated, it applies to any reference template sequencethat is herein defined as consisting of a SEQ ID NO.

Said at least two A6-targeted primers can, for example, be at least oneof SEQ ID NO: 30-34 (forward primer) and at least one of SEQ ID NO:35-37 (reverse primer).

As illustrated by the examples below (see e.g., Table 23), preferredcombinations of forward and reverse primers are as follows:

TABLE 1 (A6 primer systems): SEQ ID NO: 35 36 37 30 X X 31 X X 32 X X X33 X X 34 Xwherein X indicates that the primers can be combined with each other asa pair.

According to an advantageous embodiment of the present invention, saiddetermination of whether at least one amplicon is produced by said atleast one A6-targeted primer system, can be carried out by using inreal-time amplification at least one probe, preferably at least oneA6-targeted probe.

Advantageously, said determination of whether at least one amplicon isproduced is carried out by using in real-time amplification at least oneA6-targeted probe, which consists of one of SEQ ID NO:38-40, or of oneof the complementary sequences thereof (i.e., sequences of the samelength, which are fully complementary over the entire length of thesequence), and optionally at least one 5′ and/or 3′ detection labeland/or at least one HPV-unrelated arm intended to carry a quencher or areporter (e.g., a fluorophore), such as at least one beacon arm, orScorpion™ arm, preferably at least one of such arms in 5′ and/or 3′,most preferably two of such arms, in 5′ and in 3′, respectively.

As illustrated by the examples below (see e.g., table 23), preferredcombinations of primer pair and probe are as follows:

TABLE 2 (A6 primer and probe systems): A6 Primer pair At least one probeamplification system SEQ ID NO: of SEQ ID NO: A 30; 35 38 AE 30; 37 38and/or 40 B 31; 35 38 BE 31; 37 38 and/or 40 C 32; 36 39 CA 32; 35 38and/or 39 CE 32; 37 38 and/or 39 and/or 40 D 33; 35 38 DE 33; 37 38and/or 40 E 34; 37 40

Advantageously, said at least one A6-targeted probe is a beacon probe,the sequence of which is one of SEQ ID NO:41-45, or of one of thecomplementary sequences thereof (i.e., sequences of the same length,which are fully complementary over the entire length of the sequence).

As illustrated by the examples below (see e.g., Table 23), mostpreferred combinations of primer pair and beacon probes are as follows:

TABLE 3 (A6 primer and beacon probe systems): A6 amplification Primerpair SEQ ID At least one beacon probe system NO: of SEQ ID NO: A 30; 3541 AE 30; 37 41 and/or 44 and/or 45 B 31; 35 41 BE 31; 37 41 and/or 44and/or 45 C 32; 36 42 and/or 43 CA 32; 35 41 and/or 42 and/or 43 CE 32;37 41 and/or 42 and/or 43 and/or 44 and/or 45 D 33; 35 41 DE 33; 37 41and/or 44 and/or 45 E 34; 37 44 and/or 45

Preferably, the respective sequences of said at least two A5-targetedprimers are suitable for use in the amplification of at least onereference template sequence, which is a fragment consisting of positions678-902 (SEQ ID NO:326) of the HPV51 sequence of SEQ ID NO:421(accession NC_(—)001533.1), or a conservative sub-fragment thereof,which has retained the property of being a suitable reference templatesequence, to construct and produce A5-targeted primers, which allow fora real-time multiplex detection of those HPV, which can be oncogenic forthe mucosal epithelia, preferably of at least the five most common HPV(HPV16, 18, 45, 31, 33), still preferably at least 7 HR HPV, even stillpreferably the five most common HR HPV as well as at least two other HRHPV, advantageously at least two other HR HPV belonging to groups A6and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), more preferably atleast the 13 HR HPV, most preferably at least the 13 HR HPV and at leastone, at least two, at least three, at least four, or the five of HPV66,53, 82, 67, 85; or a sequence which is fully complementary to saidfragment or sub-fragment over the entire length of said fragment orsub-fragment.

More preferably, the respective sequences of said at least twoA5-targeted primers are suitable for use in the specific amplificationof at least one reference template sequence, wherein said at least onereference template sequence consists of one of SEQ ID NO: 1-5 and NO:320-333, as shown in Table 12; or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID sequence.

Said reference template sequences share the specific technical featureof being suitable references to construct and produce A5-targetedprimers, which allow for a real-time multiplex detection of HPV, whichcan be oncogenic for the mucosal epithelia, preferably of at least thefive most common HPV (HPV16, 18, 45, 31, 33), still preferably at least7 HR HPV, even still preferably the five most common HR HPV as well asat least two other HR HPV, advantageously at least two other HR HPVbelonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18),more preferably at least the 13 HR HPV, most preferably at least the 13HR HPV and at least one, at least two, at least three, at least four, orthe five of HPV66, 53, 82, 67, 85, and preferably for a real-timequantitative multiplex detection of such HPV.

Said at least two A5-targeted primers may, for example, be at least oneof SEQ ID NO: 6-10 (forward primer) and at least one of SEQ ID NO: 11-15(reverse primer).

As illustrated by the examples below (see e.g., Table 17), preferredcombinations of forward and reverse primers are as follows:

TABLE 4 (A5 primer systems): SEQ ID NO: 6 7 8 9 10 11 X X X X X 12 X X XX X 13 X 14 X X X X X 15 X X Xwherein X indicates that the primers can be combined with each other asa pair.

Advantageously, said determination of whether at least one amplicon isproduced is carried out by using in real-time amplification at least oneA5-targeted probe, which consists of one of SEQ ID NO:16-19, or of oneof the complementary sequences thereof (i.e., sequences of the samelength, which are fully complementary over the entire length of thesequence), and optionally at least one detection label and/or at leastone HPV-unrelated arm intended to carry a quencher or a reporter (e.g.,a fluorophore), such as at least one beacon arm, or Scorpion™ arm,preferably at least one of such arms in 5′ and/or 3′, most preferablytwo of such arms, in 5′ and in 3′, respectively.

As illustrated by the examples below (see e.g., Table 17), preferredcombinations of primer pair and probe are as follows:

TABLE 5 (A5 primer and probe systems): A5 amplification Primer pair SEQID At least one probe system NO: of SEQ ID NO: A 6; 11 16 AB 6; 12 16 AD6; 14 16 B 7; 12 16; 17 BA 7; 11 16; 17 BD 7; 14 16; 17 C 8; 13 18 CA 8;11 16; 17; 18; 19 CB 8; 12 16; 17; 18; 19 CD 8; 14 16; 17; 18; 19 CE 8;15 18; 19 D 9; 14 16; 17; 19 DA 9; 11 16; 17; 19 DB 9; 12 16; 17; 19 DE9; 15 19 E 10; 15  19 EA 10; 11  16; 17; 19 EB 10; 12  16; 17; 19 ED 10;14  16; 17; 19

Advantageously, said at least one A5-targeted probe is a beacon probe,the sequence of which is one of SEQ ID NO: 20-24, or of one of thecomplementary sequences thereof (i.e., sequences of the same length,which are fully complementary over the entire length of the sequence).

As illustrated by the examples below (see e.g., Table 17), mostpreferred combinations of primer pair and probe are as follows:

TABLE 6 (A5 primer and beacon probe systems): A5 amplification Primerpair SEQ ID At least one beacon probe system NO: of SEQ ID NO: A 6; 1120; 21 AB 6; 12 20; 21 AD 6; 14 20; 21 B 7; 12 20; 21; 22 BA 7; 11 20;21; 22 BD 7; 14 20; 21; 22 C 8; 13 23 CA 8; 11 20; 21; 22; 23; 24 CB 8;12 20; 21; 22; 23; 24 CD 8; 14 20; 21; 22; 23; 24 CE 8; 15 23; 24 D 9;14 20; 21; 22; 24 DA 9; 11 20; 21; 22; 24 DB 9; 12 20; 21; 22; 24 DE 9;15 24 E 10; 15  24 EA 10; 11  20; 21; 22; 24 EB 10; 12  20; 21; 22; 24ED 10; 14  20; 21; 22; 24

Preferably, the respective sequences of said at least two A9-targetedprimers are suitable for use in the amplification of at least onereference template sequence, which is:

-   -   a fragment consisting of positions 2707-2794 (SEQ ID NO:122) of        the HPV16 sequence of SEQ ID NO:422 (accession NC_(—)001526.1);        or a conservative sub-fragment thereof, which has retained the        property of being a suitable reference template sequence, to        construct and produce A9-targeted primers, which allow for a        real-time multiplex detection of those HPV, which can be        oncogenic for the mucosal epithelia, preferably of at least the        five most common HPV (HPV16, 18, 45, 31, 33), still preferably        at least 7 HR HPV, even still preferably the five most common HR        HPV as well as at least two other HR HPV, advantageously at        least two other HR HPV belonging to groups A6 and/or A5 (e.g.,        HPV 56, 51, 33, 31, 16, 45, 18), more preferably at least the 13        HR HPV, most preferably at least the 13 HR HPV and at least one,        at least two, at least three, at least four, or the five of        HPV66, 53, 82, 67, 85; or a sequence which is fully        complementary to said fragment or sub-fragment over the entire        length of said fragment or sub-fragment or    -   a fragment consisting of positions 3600-3840 (SEQ ID NO:377) of        the HPV16 sequence of SEQ ID NO:422 (accession NC_(—)001526.1);        or a conservative sub-fragment thereof, which has retained the        property of being a suitable reference template sequence, to        construct and produce A9-targeted primers, which allow for a        real-time multiplex detection of those HPV, which can be        oncogenic for the mucosal epithelia, preferably of at least the        five most common HPV (HPV16, 18, 45, 31, 33), still preferably        at least 7 HR HPV, even still preferably the five most common HR        HPV as well as at least two other HR HPV, advantageously at        least two other HR HPV belonging to groups A6 and/or A5 (e.g.,        HPV 56, 51, 33, 31, 16, 45, 18), more preferably at least the 13        HR HPV, most preferably at least the 13 HR HPV and at least one,        at least two, at least three, at least four, or the five of        HPV66, 53, 82, 67, 85; or a sequence which is fully        complementary to said fragment or sub-fragment over the entire        length of said fragment or sub-fragment.

More preferably, the respective sequences of said at least twoA9-targeted primers are suitable for use in the specific amplificationof at least one reference template sequence, which consists of any oneof SEQ ID NO: 122-210 and 359-419, as shown in Table 30; or a sequencewhich is fully complementary thereto over the entire length of said SEQID NO sequence.

Said reference template sequences notably share the specific technicalfeature of being suitable references to construct and produceA9-targeted primers, which allow for a real-time multiplex detection ofHPV, which can be oncogenic for the mucosal epithelia, preferably of atleast the five most common HPV (HPV16, 18, 45, 31, 33), still preferablyat least 7 HR HPV, even still preferably the five most common HR HPV aswell as at least two other HR HPV, advantageously at least two other HRHPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45,18), more preferably at least the 13 HR HPV, most preferably at leastthe 13 HR HPV and at least one, at least two, at least three, at leastfour, or the five of HPV66, 53, 82, 67, 85, and preferably for areal-time quantitative multiplex detection of such HPV.

Said at least two A9-targeted primers can, for example, be at least oneof SEQ ID NO: 211-239 (forward primers) and at least one of SEQ ID NO:240-265 (reverse primers).

As illustrated by the examples below (see e.g., Table 35), preferredcombinations of forward and reverse primers are as follows:

-   -   A9 amplification system C: at least one of SEQ ID NO:211-217 and        at least one of SEQ ID NO:240-241; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least three of SEQ ID NO: 211-217, e.g., 212, 214 and 216, and        both of SEQ ID NO:240-241;    -   A9 amplification system E1: at least one of SEQ ID NO:218-220        and at least one of SEQ ID NO:242-247; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:218-220 and at least five of SEQ ID        NO:242-247 (e.g., SEQ ID NO:242-243, 245-247);    -   A9 amplification system E2: at least one of SEQ ID NO:221-223        and at least one of SEQ ID NO:242-247; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:221-223 and at least five of SEQ ID        NO:242-247 (e.g., SEQ ID NO:242-243, 245-247);    -   A9 amplification system E3: at least one of SEQ ID NO:221-223        and at least one of SEQ ID NO:248-255; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:221-223 and at least five of SEQ ID        NO:248-255 (e.g., SEQ ID NO:248-252);    -   A9 amplification system E4: at least one of SEQ ID NO:224-226        and at least one of SEQ ID NO:248-255; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three: of SEQ ID NO:224-226 and at least five of SEQ        ID NO:248-255 (e.g., SEQ ID NO:248-252);    -   A9 amplification system E5: at least one of SEQ ID NO:224-226        and at least one of SEQ ID NO:242-247; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:224-226 and at least five of SEQ ID        NO:242-247 (e.g., SEQ ID NO:242-243, 245-247);    -   A9 amplification system E6: at least one of SEQ ID NO:218-220        and at least one of SEQ ID NO:248-255; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:218-220 and at least five of SEQ ID        NO:248-255 (e.g., SEQ ID NO:248-252);    -   A9 amplification system E1H Z7: at least one of SEQ ID        NO:218-220 and at least one of SEQ ID NO:256-259; 261-265;        preferably, when amplification of all oncogenic HPV of group A9        is desired, at least the three of SEQ ID NO:218-220 and at least        four of SEQ ID NO: 256-259; 261-265 (e.g., SEQ ID NO:258; 261;        264; 265);    -   A9 amplification system E1H Z8: at least one of SEQ ID NO:        218-220 and at least one of SEQ ID NO:256-259; 261-265;        preferably, when amplification of all oncogenic HPV of group A9        is desired, at least the three of SEQ ID NO:218-220 and at least        four of SEQ ID NO:256-259; 261-265 (e.g., SEQ ID NO:258; 261;        264; 265);    -   A9 amplification system E2H Z7: at least one of SEQ ID        NO:221-223 and at least one of SEQ ID NO:256-259; 261-265;        preferably, when amplification of all oncogenic HPV of group A9        is desired, at least the three of SEQ ID NO:221-223 and at least        four of SEQ ID NO:256-259; 261-265 (e.g., SEQ ID NO: 258; 261;        264; 265);    -   A9 amplification system E2H Z8: identical to A9 amplification        system E2H Z7, i.e., at least one of SEQ ID NO:221-223 and at        least one of SEQ ID NO: 256-259; 261-265; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:221-223 and at least four of SEQ ID        NO:256-259; 261-265 (e.g., SEQ ID NO:258; 261; 264; 265);    -   A9 amplification system E4H Z7: at least one of SEQ ID        NO:224-226 and at least one of SEQ ID NO: 256-259; 261-265;        preferably, when amplification of all oncogenic HPV of group A9        is desired, at least the three of SEQ ID NO:224-226 and at least        four of SEQ ID NO:256-259; 261-265 (e.g., SEQ ID NO:258; 261;        264; 265);    -   A9 amplification system E4H Z8: at least one of SEQ ID        NO:224-226 and at least one of SEQ ID NO: 256-259; 261-265;        preferably, when amplification of all oncogenic HPV of group A9        is desired, at least the three of SEQ ID NO:224-226 and at least        four of SEQ ID NO:256-259; 261-265 (e.g., SEQ ID NO: 258; 261;        264; 265);    -   A9 amplification system F: at least one of SEQ ID NO:227-230 and        at least one of SEQ ID NO:248-255; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the four of SEQ ID NO:227-230 and at least five of SEQ ID        NO:248-255 (e.g., SEQ ID NO:248-252);    -   A9 amplification system FE: at least one of SEQ ID NO:227-230        and at least one of SEQ ID NO:242-247; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the four of SEQ ID NO:227-230 and at least five of SEQ ID        NO:242-247 (e.g., SEQ ID NO:242-243; 245-247);    -   A9 amplification system FH Z7: at least one of SEQ ID NO:227-230        and at least one of SEQ ID NO:256-259; 261-265; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the four of SEQ ID NO:227-230 and at least four of SEQ ID        NO:256-259; 261-265 (e.g., SEQ ID NO:258; 261; 264; 265);    -   A9 amplification system FHZ8: identical to A9 amplification        system FH Z7, i.e., at least one of SEQ ID NO:227-230 and at        least one of SEQ ID NO:256-259; 261-265; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:227-230 and at least four of SEQ ID        NO:256-259; 261-265 (e.g., SEQ ID NO:258; 261; 264; 265);    -   A9 amplification system G Z7: at least one of SEQ ID NO:227-230        and at least one of SEQ ID NO:256-261; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least the three of SEQ ID NO:227-230 and at least five of SEQ ID        NO:256-261 (e.g., SEQ ID NO:257-261);    -   A9 amplification system G Z8: identical to A9 amplification        system G Z7, i.e., at least one of SEQ ID NO:227-230 and at        least one of SEQ ID NO:256-261; preferably, when amplification        of all oncogenic HPV of group A9 is desired, at least the three        of SEQ ID NO:227-230 and at least five of SEQ ID NO:256-261        (e.g., SEQ ID NO:257-261);    -   A9 amplification system H: at least one of SEQ ID NO:231-239 and        at least one of SEQ ID NO: 256-259; 261-265; preferably, when        amplification of all oncogenic HPV of group A9 is desired, at        least three of SEQ ID NO:231-239 (e.g., SEQ ID NO:232; 234; 235)        and at least four of SEQ ID NO: 256-259; 261-265 (e.g., SEQ ID        NO:258; 261; 264; 265).

Advantageously, said determination of whether at least one amplicon isproduced is carried out by using in real-time amplification at least oneA9-targeted probe, which consists of one of SEQ ID NO: 266-282, or ofone of the complementary sequences thereof (i.e., sequences of the samelength, which are fully complementary over the entire length of thesequence), and optionally at least one detection label and/or at leastone HPV-unrelated arm intended to carry a quencher or a reporter (e.g.,a fluorophore), such as at least one beacon arm, or Scorpion™ arm,preferably at least one of such arms in 5′ and/or 3′, most preferablytwo of such arms, in 5′ and in 3′, respectively.

As illustrated by the examples below (see e.g., Table 35), preferredcombinations of primer pair and probe are as follows:

TABLE 7 (A9 primer and probe systems): A9 amplification system At leastone probe of SEQ ID NO: C 266-271 E1 272-276 E2 272-276 E3 272-276 E4272-276 E5 272-276 E6 272-276 E1H Z7 272-276 E1H Z8 277-282 E2H Z7272-276 E2H Z8 277-282 E4H Z7 272-276 E4H Z8 277-282 F 272-276 FE272-276 FH Z7 272-276 FH Z8 277-282 G Z7 272-276 G Z8 277-282 H 277-282

Advantageously, said at least one A9-targeted probe is a beacon probe,the sequence of which is one of SEQ ID NO: 283-319, or of one of thecomplementary sequences thereof (i.e., sequences of the same length,which are fully complementary over the entire length of the sequence).

As illustrated by the examples below (see e.g., Table 35), mostpreferred combinations of primer pair and probe are as follows:

TABLE 8 (A9 primer and beacon probe systems): A9 amplification system Atleast one beacon probe of SEQ ID NO: C 283-295 E1 296-303 E2 296-303 E3296-303 E4 296-303 E5 296-303 E6 296-303 E1H Z7 296-303 E1H Z8 304-319E2H Z7 296-303 E2H Z8 304-319 E4H Z7 296-303 E4H Z8 304-319 F 296-303 FE296-303 FH Z7 296-303 FH Z8 304-319 G Z7 296-303 G Z8 304-319 H 304-319

Some of the specificity results of the above-mentioned probes are shownin Tables 60-68 (Specificity A9).

In all tables, when the name of a probe differs from the name of anotherprobe by only the last letter (e.g., A9E1S10 and A9E1S10a), these probeshave the same hybridizing segment, and only differ in their beacon arms.

Gray boxes indicate that the tested probe detects the amplicon.

For example, probe A9E1S11 of SEQ ID NO: 285 detects HPV31 and HPV35,without detecting the other HPV.

For example, probe A9E1S10a of SEQ ID NO: 284 detects all oncogenic HPVof group A9 (HPV16, 31, 33, 35, 52, 58, 67) and one HPV of group A6(HPV53), and probe A9E1S12a of SEQ ID NO: 288 detects HPV31 and HPV35,without detecting HPV53 (“ND”). Hence, a combination of A9E1S10a and ofA9E1S12a allows the specific detection of HPV16, 31, 33, 35, 52, 58, 67,53.

Any combination that the skilled person may find appropriate is hereinspecifically encompassed.

Preferably, the respective sequences of said at least two A7-targetedprimers are suitable for use in the amplification of at least onereference template sequence, wherein said at least one referencetemplate sequence is:

-   -   a fragment consisting of positions 1895-2103 (SEQ ID NO:48) of        the HPV18 sequence of SEQ ID NO:423 (accession NC_(—)001357.1);        or a conservative sub-fragment thereof, which has retained the        property of being a suitable reference template sequence, to        construct and produce A7-targeted primers, which allow for a        real-time multiplex detection of those HPV, which can be        oncogenic for the mucosal epithelia, preferably of at least the        five most common HPV (HPV16, 18, 45, 31, 33), still preferably        at least 7 HR HPV, even still preferably the five most common HR        HPV as well as at least two other HR HPV, advantageously at        least two other HR HPV belonging to groups A6 and/or A5 (e.g.,        HPV 56, 51, 33, 31, 16, 45, 18), more preferably at least the 13        HR HPV, most preferably at least the 13 HR HPV and at least one,        at least two, at least three, at least four, or the five of        HPV66, 53, 82, 67, 85; or a sequence which is fully        complementary to said fragment or sub-fragment over the entire        length of said fragment or sub-fragment, or    -   a fragment consisting of positions 916-1044 (SEQ ID NO: 65) of        the HPV18 sequence of SEQ ID NO:423 (accession NC_(—)001357.1);        or a conservative sub-fragment thereof, which has retained the        property of being a suitable reference template sequence, to        construct and produce A7-targeted primers, which allow for a        real-time multiplex detection of those HPV, which can be        oncogenic for the mucosal epithelia, preferably of at least the        five most common HPV (HPV16, 18, 45, 31, 33), still preferably        at least 7 HR HPV, even still preferably the five most common HR        HPV as well as at least two other HR HPV, advantageously at        least two other HR HPV belonging to groups A6 and/or A5 (e.g.,        HPV 56, 51, 33, 31, 16, 45, 18), more preferably at least the 13        HR HPV, most preferably at least the 13 HR HPV and at least one,        at least two, at least three, at least four, or the five of        HPV66, 53, 82, 67, 85; or a sequence which is fully        complementary to said fragment or sub-fragment over the entire        length of said fragment or sub-fragment.

More preferably, the respective sequences of said at least twoA7-targeted primers are suitable for use in the specific amplificationof at least one reference template sequence, which consists of one ofSEQ ID NO:46-67; 339-358, as shown in Table 24; or a sequence which isfully complementary thereto over the entire length of said SEQ ID NOsequence.

Said reference template sequences notably share the specific technicalfeature of being suitable references to construct and produceA7-targeted primers, which allow for a real-time multiplex detection ofHPV, which can be oncogenic for the mucosal epithelia, preferably of atleast the five most common HPV (HPV16, 18, 45, 31, 33), still preferablyat least 7 HR HPV, even still preferably the five most common HR HPV aswell as at least two other HR HPV, advantageously at least two other HRHPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45,18), more preferably at least the 13 HR HPV, most preferably at leastthe 13 HR HPV and at least one, at least two, at least three, at leastfour, or the five of HPV66, 53, 82, 67, 85, and preferably for areal-time quantitative multiplex detection of such HPV.

Said at least two A7-targeted primers can, for example, be at least oneof SEQ ID NO: 68-78 (forward primer) and at least one of SEQ ID NO:79-87 (reverse primer).

As illustrated by the examples below (see e.g., Table 29), preferredcombinations of forward and reverse primers are as follows:

-   -   A7 amplification system A: at least one of SEQ ID NO:68-70 and        at least one of SEQ ID NO:79-81; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system A1: identical to A7 amplification system        A, i.e., at least one of SEQ ID NO:68-70 and at least one of SEQ        ID NO:79-81; preferably, when amplification of all oncogenic HPV        of group A7 is desired, all of them;    -   A7 amplification system A2: identical to A7 amplification system        A or A1, i.e., at least one of SEQ ID NO:68-70 and at least one        of SEQ ID NO:79-81; preferably, when amplification of all        oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system AB: at least one of SEQ ID NO:68-70 and        at least one of SEQ ID NO:82-83; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system AC1: at least one of SEQ ID NO:68-70 and        at least one of SEQ ID NO:84-85; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system AC2: identical to A7 amplification        system AC1, i.e., at least one of SEQ ID NO:68-70 and at least        one of SEQ ID NO:84-85; preferably, when amplification of all        oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system AC3: identical to A7 amplification        system AC1 or AC2, i.e., at least one of SEQ ID NO:68-70 and at        least one of SEQ ID NO:84-85; preferably, when amplification of        all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system B: at least one of SEQ ID NO:71-73 and        at least one of SEQ ID NO:82-83; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system BA1: at least one of SEQ ID NO:71-73 and        at least one of SEQ ID NO:79-81; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system BA2: identical to A7 amplification        system BA1, i.e., at least one of SEQ ID NO:71-73 and at least        one of SEQ ID NO:79-81; preferably, when amplification of all        oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system BA3: identical to A7 amplification        system BA1 or BA2, i.e., at least one of SEQ ID NO:71-73 and at        least one of SEQ ID NO:79-81; preferably, when amplification of        all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system BC1: at least one of SEQ ID NO:71-73 and        at least one of SEQ ID NO:8485; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system BC2: identical to A7 amplification        system BC1, i.e., at least one of SEQ ID NO:71-73 and at least        one of SEQ ID NO:84-85; preferably, when amplification of all        oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system BC3: identical to A7 amplification        system BC1 or BC2, i.e., at least one of SEQ ID NO:71-73 and at        least one of SEQ ID NO:8485; preferably, when amplification of        all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system C: at least one of SEQ ID NO:74-76 and        at least one of SEQ ID NO:8485; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system C1: identical to A7 amplification system        C, i.e., at least one of SEQ ID NO:74-76 and at least one of SEQ        ID NO:84-85; preferably, when amplification of all oncogenic HPV        of group A7 is desired, all of them;    -   A7 amplification system CA1: identical to A7 amplification        system C1, i.e., at least one of SEQ ID NO:74-76 and at least        one of SEQ ID NO:79-81; preferably, when amplification of all        oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system CA2: identical to A7 amplification        system C1 or CA1, i.e., at least one of SEQ ID NO:7476 and at        least one of SEQ ID NO:79-81; preferably, when amplification of        all oncogenic HPV of group A7 is desired, all of them;    -   A7 amplification system D: at least one of SEQ ID NO:77-78 and        at least one of SEQ ID NO:86-87; preferably, when amplification        of all oncogenic HPV of group A7 is desired, all of them.

Advantageously, said determination of whether at least one amplicon isproduced is carried out by using in real-time amplification at least oneA7-targeted probe, which consists of one of SEQ ID NO:88-101, or of oneof the complementary sequences thereof (i.e., sequences of the samelength, which are fully complementary over the entire length of thesequence), and optionally at least one detection label and/or at leastone HPV-unrelated arm intended to carry a quencher or a reporter (e.g.,a fluorophore), such as at least one beacon arm, or Scorpion™ arm,preferably at least one of such arms in 5′ and/or 3′, most preferablytwo of such arms, in 5′ and in 3′, respectively.

As illustrated by the examples below (see e.g., Table 29), preferredcombinations of primer pair and probe are as follows:

TABLE 10 (A7 primer and probe systems): A7 amplification At least oneprobe system of SEQ ID NO: A 88-92 A1 92-95 A2 89-91; 96 AB 92-95 AC188-92 AC2 92-95 AC3 89-91; 96 B 92-95 BA1 88-92 BA2 92-95 BA3 89-91; 96BC1 88-92 BC2 92-95 BC3 89-91; 96 C 89-91; 96 C1 88-91; 96 CA1 89-91; 96CA2 88-91; 96 D 97-101

Advantageously, said at least one A7-targeted probe is a beacon probe,the sequence of which is one of SEQ ID NO:102-121, or of one of thecomplementary sequences thereof (i.e., sequences of the same length,which are fully complementary over the entire length of the sequence).

As illustrated by the examples below (see e.g., Table 29), mostpreferred combinations of primer pair and probe are as follows:

TABLE 11 (A7 primer and beacon probe systems): A7 amplification At leastone beacon system probe of SEQ ID NO: A 102-106 A1 106-110 A2 105;111-114 AB 106-110 AC1 102-106 AC2 106-110 AC3 105; 111-114 B 106-110BA1 102-106 BA2 106-110 BA3 105; 111-114 BC1 102-106 BC2 106-110 BC3105; 111-114 C 105; 111-114 C1 102-105; 114 CA1 105; 111-114 CA2102-105; 114 D 115-121

Said amplification can be any nucleic acid amplification, which is foundappropriate to the skilled person, for example a PCR (Polymerase ChainReaction), or an isothermal amplification technique, e.g., TMA(transcription mediated amplification), NASBA (nucleic acid sequencebased amplification), 3SR (self sustained sequence replication) orstrand displacement amplification. Said amplification preferably is aPCR.

In a preferred embodiment, the primers according to the invention areused in a final concentration range 100-200 nM. Typically, said primerscan be used at a final concentration range 200-1500 nM, preferably250-1000 nM, more preferably 500-1000 nM, even more preferably 600-1000nM.

Probe concentration in a PCR reaction can be optimized, typically byvarying the final concentration from 50 nM to 1000 nM. In a preferredembodiment, the probes according to the invention are used at a finalconcentration range 50-1000 nM, preferably 100-800 nM, more preferably100-600 nM, even more preferably 200-600 nM.

Appropriate amplification conditions are known to those skilled in theart. They include temperature conditions, in particular thermal cyclingconditions, e.g. temperature, duration, number, heating rate of thecycles. In a preferred embodiment, said temperature conditions includeconditions suitable for a PCR. In another preferred embodiment, saidconditions include conditions suitable for a Q-PCR.

Any megaplex, i.e., multi-multiplex comprising at least A6-targeted onereal-time amplification system of the invention and at least oneA5-targeted one real-time amplification system of the invention and atleast one A9-targeted one real-time amplification system of theinvention and at least one A7-targeted one real-time amplificationsystem of the invention, which would be contemplated by the person ofordinary skill in the art is encompassed by the present application.

For example, the A5-targeted system E, and the A6-targeted system A, andthe A7-targeted system A, and the A9-targeted system H, can be usedtogether in a single-tube assay, thereby forming a megaplex (“megaplexEAAH”).

For example, the A5-targeted system E, and the A6-targeted system B, andthe A7-targeted system A, and the A9-targeted system C, can be usedtogether in a single-tube assay, thereby forming a megaplex (“megaplexEBAC”).

Illustrative specificity and sensitivity results of such megaplex areshown in tables 83-88 below.

From tables 84 and 87 (“megaplex EAAH”), it can be seen that such amegaplex enable to efficiently detect the oncogenic HPV, namely the 13HR HPV and five other oncogenic HPV (HPV66, 53, 82, 67, 85), and thatthis megaplex has sufficiently homogeneous sensitivity and specificity(Ct) results to be quantitative for at least the 13 HR HPV and fourother oncogenic HPV (HPV66, 53, 82, 85). From tables 86 and 88(“megaplex EBAC”), it can be seen that such a megaplex enable toefficiently detect the oncogenic HPV, namely at least the 13 HR HPV, andthat this megaplex has sufficiently homogeneous sensitivity andspecificity (Ct) results to be quantitative for at least the five mostcommon HR HPV (HPV16, 18, 45, 31, 33).

Preferred megaplex notably comprise the above-mentioned EAAH and EBACmegaplex, as well as the following megaplex systems:

-   -   the combination of A5-targeted system E, and the A6-targeted        system B, and the A7-targeted system C, and the A9-targeted        system C, can be used together in a single-tube assay, thereby        forming a megaplex (“megaplex EBCC”);    -   the combination of A5-targeted system E, and the A6-targeted        system B, and the A7-targeted system B, and the A9-targeted        system C, can be used together in a single-tube assay, thereby        forming a megaplex (“megaplex EBBC”);    -   the combination of A5-targeted system E, and the A6-targeted        system B, and the A7-targeted system D, and the A9-targeted        system C, can be used together in a single-tube assay, thereby        forming a megaplex (“megaplex EBDC”);    -   the combination of A5-targeted system E, and the A6-targeted        system B, and the A7-targeted system A, and the A9-targeted        system H, can be used together in a single-tube assay, thereby        forming a megaplex (“megaplex EBAH”).

The present application also relates to any amplicon obtainable byimplementation of the process according to any one claims 1-45 on aHPV-containing sample, which contains at least one HPV of group A6, A5,A9 or A7, for example, a sample which contains HPV66 and/or HPV53 and/orHPV82 and/or HPV58 and/or HPV33 and/or HPV67 and/or HPV52 and/or HPV35and/or HPV31 and/or HPV68 and/or HPV39 and/or HPV85 and/or HPV59 and/orHPV45.

The invention is also directed to a polynucleotide suitable for use as areference template sequence in the design of primers that can be used inmultiplex to cover at least the five most common HR HPV (HPV16, 18, 45,31, 33),

preferably at least 7 HR HPV, still preferably the five most common HRHPV as well as at least two other HR HPV, advantageously at least twoother HR HPV belonging to groups A6 and/or A5 (e.g., HPV 56, 51, 33, 31,16, 45, 18),even still preferably at least the thirteen HPV known as HR HPV (HPVtypes 56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59, 45 and 18),more preferably, at least the thirteen HPV as well as at least one amongHPV types 66, 82, 67, 85, and 53still more preferably, at least the thirteen HPV as well as at least twoamong HPV types 66, 82, 67, 85, and 53even still more preferably, at least the thirteen HPV as well as atleast three among HPV types 66, 82, 67, 85, and 53most preferably, at least the thirteen HPV as well as at least fouramong HPV types 66, 82, 67, 85, and 53, notably of least the seventeenmucosal HPV, consisting of said 13 HR HPV and HPV types 66, 82, 67, 85,still most preferably, at least the thirteen HPV as well as at least thefive HPV types 66, 82, 67, 85, and 53,in a single amplification run while still offering a real timequantitative amplification thereof.

Of course, the polynucleotides according to the present invention arealso suitable for further protocols, including simplex protocols,multiplex protocols, end-point protocols, qualitative protocols,quantitative protocols, combinations thereof, and the like.

By polynucleotide, we hereby understand any polymer of nucleotides,wherein nucleotides can be ribonucleotides, deoxyribonucleotides,dideoxyribonucleotides, degenerated nucleotides, and the like. Saidnucleotides are preferably single-stranded, but can also be doublestranded. The length of said polynucleotides can vary, and is usuallyunder 500 nucleotides (nt), preferably in the range of 50-400 nt, morepreferably 100-300 nt, even more preferably 80-260 nt.

The present application also relates to any polynucleotide suitable foruse as a reference template sequence in the design of primers that canbe used in a single-tube multiplex to amplify those HPV of groups A6,A5, A9 and A7, and in the design of probes that can be used in saidsingle-tube multiplex for real-time detection of said amplified HPV,said reference template polynucleotide being selected from:

-   -   for group A6: a fragment consisting of positions 413-791 of the        HPV56 sequence of SEQ ID NO:420 (accession NC_(—)001594.1), or a        conservative sub-fragment thereof, or a sequence which is fully        complementary to said fragment or sub-fragment over the entire        length of said fragment or sub-fragment;    -   for group A5: a fragment consisting of positions 678-902 of the        HPV51 sequence of SEQ ID NO:421 (accession NC_(—)001533.1), or a        conservative sub-fragment thereof, or a sequence which is fully        complementary to said fragment or sub-fragment over the entire        length of said fragment or sub-fragment;    -   for group A9: a fragment consisting of positions 2707-2794 of        the HPV16 sequence of SEQ ID NO:422 (accession NC_(—)001526.1),        or a conservative sub-fragment thereof, or a sequence which is        fully complementary to said fragment or sub-fragment over the        entire length of said fragment or sub-fragment; or a fragment        consisting of positions 3600-3840 of the HPV16 sequence of SEQ        ID NO:422 (accession NC_(—)001526.1), or a conservative        sub-fragment thereof, or a sequence which is fully complementary        to said fragment or sub-fragment over the entire length of said        fragment or sub-fragment;    -   for group A7: a fragment consisting of positions 1895-2103 of        the HPV18 sequence of SEQ ID NO:423 (accession NC_(—)001357.1),        or a conservative sub-fragment thereof, or a sequence which is        fully complementary to said fragment or sub-fragment over the        entire length of said fragment or sub-fragment; or a fragment        consisting of positions 916-1044 of the HPV18 sequence of SEQ ID        NO:423 (accession NC_(—)001357.1), or a conservative        sub-fragment thereof, or a sequence which is fully complementary        to said fragment or sub-fragment over the entire length of said        fragment or sub-fragment,        wherein said conservative fragment thereof have retained the        property of being a suitable reference template sequence, to        construct and produce group-targeted primers, which allow for a        real-time multiplex detection of those HPV, which can be        oncogenic for the mucosal epithelia.

The present application more particularly relates to any referencetemplate polynucleotide, which consists of one of SEQ ID NO:25-29 andNO:334-338 (group A6-targeted reference template polynucleotides), or asequence which is fully complementary thereto over the entire length ofsaid SEQ ID NO sequence.

The present application more particularly relates to any referencetemplate polynucleotide, which consists of one of SEQ ID NO: 1-5 andNO:320-333 (group A5-targeted reference template sequences), or asequence which is fully complementary thereto over the entire length ofsaid SEQ ID NO sequence.

The present application more particularly relates to any referencetemplate polynucleotide, which consists of one of SEQ ID NO: SEQ IDNO:122-210 and 359-419 (group A9-targeted reference template sequences),or a sequence which is fully complementary thereto over the entirelength of said SEQ ID NO sequence.

The present application more particularly relates to any referencetemplate polynucleotide, which consists of one of SEQ ID NO: 46-67;339-358 (group A7-targeted reference template sequences), or a sequencewhich is fully complementary thereto over the entire length of said SEQID NO sequence.

Conservative variants of the reference template polynucleotide of theinvention are also encompassed by the present application. Conservativevariants of a given parent reference template polynucleotide notablyinclude any polynucleotide, which derives from said parent referencetemplate polynucleotide, or the sequence which is fully complementarythereto, by deletion and/or substitution and/or addition of at least onenucleotide, but which has retained the capacity of being a referencetemplate polynucleotide for designing and building an A5- and/or A6-and/or A7- and/or A9-targeted primer pair enabling the amplification ofat least one HPV, which can be oncogenic for the mucosal epithelia.Illustrative conservative variants usually comprise thosepolynucleotides, which have a sequence identity of at least 80%,preferably of at least 85%, more preferably of at least 90%, mostpreferably of at least 95%, with said parent reference templatepolynucleotide or with the sequence which is fully complementary thereto(said identity score being computed over the entire length of saidparent reference template polynucleotide, or fully complementarysequence). Illustrative conservative variants comprise those which donot differ from plus or minus 5 nucleotides in length from the parentsequence.

The present application also relates to the primers and probes of thepresent invention, as such, i.e., as individual oligonucleotideproducts.

Conservative variants of the primers of the invention are alsoencompassed by the present application. Conservative variants of a givenparent primer notably include any oligonucleotide, which derives fromsaid parent primer by deletion and/or substitution and/or addition of atleast one nucleotide, but which has retained the capacity of being aforward or reverse A5- and/or A6- and/or A7- and/or A9-targeted primerfor the amplification of at least one HPV, which can be oncogenic forthe mucosal epithelia. Illustrative conservative variants usuallycomprise those oligonucleotides, which have a sequence identity of atleast 80%, preferably of at least 81%, more preferably of at least 83%,most preferably of least 85%, with said parent primer (said identityscore being computed over the entire length of said parent primer).Illustrative conservative variants comprise those which do not differfrom plus or minus 5 nucleotides in length from the parent sequence.

Conservative variants of the probes of the invention are alsoencompassed by the present application. Conservative variants of a givenparent probe notably include any oligonucleotide, which derives fromsaid parent probe, or the sequence which is fully complementary thereto,by deletion and/or substitution and/or addition of at least onenucleotide, but which has retained the capacity of being an A5- and/orA6- and/or A7- and/or A9-targeted probe for the detection of at leastone HPV, which can be oncogenic for the mucosal epithelia. Illustrativeconservative variants usually comprise those oligonucleotides, whichhave a sequence identity of at least 90%, preferably of at least 91%,more preferably of at least 93%, most preferably of at least 95%, withsaid parent probe or with the sequence which is fully complementarythereto (said identity score being computed over the entire length ofsaid parent probe). Illustrative conservative variants comprise thosewhich do not differ from plus or minus 5 nucleotides in length from theparent sequence.

The present application more particularly relates to primers, which aresuitable for HPV amplification, and which are especially adapted to thereal-time multiplex amplification of HPV groups A6 and A5 and A9 and A7.

Said primer can e.g., be:

-   -   an A6-targeted primer, consisting of any one of SEQ ID NO: 30-34        and SEQ ID NO: 35-37; or    -   an A5-targeted primer, consisting of any one of SEQ ID NO: 6-10        and SEQ ID NO: 11-15; or    -   an A9-targeted primer, consisting of any one of SEQ ID NO:        211-239 and SEQ ID NO: 240-265; or    -   an A7-targeted primer, consisting of any one of SEQ ID NO: 68-78        and SEQ ID NO: 79-87.

The present application more particularly relates to primer systemssuitable for HPV amplification, which are especially adapted to thereal-time multiplex amplification of HPV groups A6 and A5 and A9 and A7.

Said primer system may e.g., be:

-   -   at least one A6-targeted primer consisting of one of SEQ ID NO:        30-34, and at least one A6-targeted primer consisting of one of        SEQ ID NO: 35-37; and/or    -   at least one A5-targeted primer consisting of one of SEQ ID NO:        6-10, and at least one A5-targeted primer consisting of one of        SEQ ID NO: 11-15; and/or    -   at least one A9-targeted primer consisting of one of SEQ ID NO:        211-239, and at least one A9-targeted primer consisting of one        of SEQ ID NO: 240-265; and/or    -   at least one A7-targeted primer consisting of one of SEQ ID NO:        68-78, and at least one A7-targeted primer consisting of one of        SEQ ID NO: 79-87.

The present application more particularly relates to probes, which aresuitable for HPV detection, and which are especially adapted to thereal-time multiplex amplification of HPV groups A6 and A5 and A9 and A7.

Said probe may e.g., be:

-   -   an A6-targeted probe, consisting of any one of SEQ ID NO:38-40,        or a sequence which is fully complementary thereto over the        entire length of said SEQ ID NO sequence; or    -   an A5-targeted probe, consisting of any one of SEQ ID NO:16-19,        or a sequence which is fully complementary thereto over the        entire length of said SEQ ID NO sequence; or    -   an A9-targeted probe, consisting of any one of SEQ ID NO:        266-282, or a sequence which is fully complementary thereto over        the entire length of said SEQ ID NO sequence; or    -   an A7-targeted probe, consisting of any one of SEQ ID NO:88-101,        or a sequence which is fully complementary thereto over the        entire length of said SEQ ID NO sequence.

Said probes can be produced in various format, e.g., including Taqman™probes (hydrolysis probes), molecular Beacons™ (beacon probes ormolecular beacon probes), and Scorpion™ probes. One of preferred formatsis the beacon format. Hence, the present application more particularlyrelates to beacon probes, which are suitable for HPV detection, andwhich are especially adapted to the real-time multiplex amplification ofHPV groups A6 and A5 and A9 and A7.

Said beacon probe may e.g., be:

-   -   an A6-targeted probe, consisting of any one of SEQ ID NO:41-45,        or a sequence which is fully complementary thereto over the        entire length of said SEQ ID NO sequence; or    -   an A5-targeted probe, consisting of any one of SEQ ID NO:20-24,        or a sequence which is fully complementary thereto over the        entire length of said SEQ ID NO sequence; or    -   an A9-targeted probe, consisting of any one of SEQ ID NO:        283-319, or a sequence which is fully complementary thereto over        the entire length of said SEQ ID NO sequence; or    -   an A7-targeted probe, consisting of any one of SEQ ID        NO:102-121, or a sequence which is fully complementary thereto        over the entire length of said SEQ ID NO sequence.

Beacon probe may further comprise a quencher and a reporter (e.g., afluorophore).

Preferably, each probe has its own reporter, whereby each probe has areporter that is different from the ones displayed by the other probes,whereby each probe can be easily distinguished from the other probes.

The present application more particularly relates to primer and probesystems, which are suitable for HPV amplification, and which areespecially adapted to the real-time multiplex amplification of HPVgroups A6 and A5 and A9 and A7.

Said primer and probe system comprises at least one primer systemaccording to the invention, and at least one probe according to theinvention.

The present application further relates to any amplicon obtainable byamplification of at least one nucleic acid from an HPV of group A6, A5,A9 or A7, by means of at least one primer system according to claim 53,for example, HPV66 and/or HPV53 and/or HPV82 and/or HPV58 and/or HPV33and/or HPV67 and/or HPV52 and/or HPV35 and/or HPV31 and/or HPV68 and/orHPV39 and/or HPV85 and/or HPV59 and/or HPV45.

An amplification composition comprising such an amplicon is alsoencompassed by the present invention.

The present invention also relates to an amplification composition, apharmaceutical composition, a biological composition, comprising atleast one primer or probe of the invention.

The present invention also relates to a kit for the diagnostic orprognostic of a cervical neoplasia or cancer, comprising:

-   -   at least one primer system according to the invention, and/or    -   at least one probe according to the invention,    -   optionally, instructions for the use thereof and/or nucleotides.

Preferably, said kit comprises at least two primer systems according tothe invention, more preferably at least three primer systems accordingto the invention, most preferably at least four primer systems accordingto the invention. Said kit comprises more than one probe, e.g. at leasttwo, at least three, at least four, at least five different probes,notably when the kit is intended to discriminate between different HPVtypes.

In the kit according to the invention, the oligonucleotides (primers,probes) can be either kept separately, or partially mixed, or totallymixed.

Said oligonucleotides can be provided under dry form, or solubilized ina suitable solvent, as judged by the skilled person. Suitable solventsinclude TE, PCR-grade water, and the like.

In a preferred embodiment, the kit according to the invention can alsocontain further reagents suitable for a PCR step.

Such reagents are known to those skilled in the art, and include water,like nuclease-free water, RNase free water, DNAse-free water, PCR-gradewater; salts, like magnesium, potassium; buffers such as Tris; enzymes,including polymerases, such as Taq, Vent, Pfu (all of them Trade-Marks),activable polymerase, and the like; nucleotides like deoxynucleotides,dideoxynucleotides, dNTPs, dATP, dTTP, dCTP, dGTP, dUTP; other reagents,like DTT and/or RNase inhibitors; and polynucleotides like polyT,polydT, and other oligonucleotides, e.g., primers.

In another preferred embodiment, the kit according to the inventioncomprises PCR controls. Such controls are known in the art, and includequalitative controls, positive controls, negative controls, internalcontrols, quantitative controls, internal quantitative controls, as wellas calibration ranges. The internal control for said PCR step can be atemplate which is unrelated to the target template in the PCR step. Suchcontrols also may comprise control primers and/or control probes. Forexample, in the case of HPV detection, it is possible to use as aninternal control, a polynucleotide chosen within a gene whose presenceis excluded in a sample originating from a human body (for example, froma plant gene), and whose size and GC content is equivalent to those fromthe target sequence.

In a preferred embodiment, the kit according to the invention containsmeans for extracting and/or purifying nucleic acid from a biologicalsample, e.g. from blood, serum, plasma. Such means are well known tothose skilled in the art.

In a preferred embodiment, the kit according to the invention containsinstructions for the use thereof. Said instructions can advantageouslybe a leaflet, a card, or the like. Said instructions can also be presentunder two forms: a detailed one, gathering exhaustive information aboutthe kit and the use thereof, possibly also including literature data;and a quick-guide form or a memo, e.g., in the shape of a card,gathering the essential information needed for the use thereof.

In a preferred embodiment, said kit is a diagnostics kit, especially anin vitro diagnostics kit, i.e., an HPV diagnostics kit.

The present invention also relates to the field of diagnostics,prognosis and drug/treatment efficiency monitoring, as above-described.

The oligonucleotides according to the present invention can be used forthe diagnostic of HPV group, types, subtypes or strains. In particular,the primers and probes according to the invention can be used for invitro typing, sub-typing, and quantification of HPV nucleic acidspresent in an in vitro sample, for instance, in a patient's cervicalsample, or in a cell culture supernatant.

The term “comprising”, which is synonymous with “including” or“containing”, is open-ended, and does not exclude additional, unrecitedelement(s), ingredient(s) or method step(s), whereas the term“consisting of” is a closed term, which excludes any additional element,step, or ingredient which is not explicitly recited. The termessentially consisting of is a partially open terms which does notexclude additional, unrecited element(s), step(s), or ingredient(s), aslong as these additional element(s), step(s) or ingredient(s) do notmaterially affect the basic and novel properties of the invention.

The term “comprising” (or “comprise(s)”) hence includes the term“consisting of” (“consist(s) of”), as well as the term “essentiallyconsisting of” (“essentially consist(s) of”). Accordingly, the term“comprising” (or “comprise(s)”) is, in the present application, meant asmore particularly encompassing the term “consisting of” (“consist(s)of”), and the term “essentially consisting of” (“essentially consist(s)of”).

In the present application, the term “at least x” relating to a set orgroup of n elements (wherein x is different from zero, and n is a numberthat is higher than x), explicitly encompasses each value, which iscomprises between x and n. For example, the term “at least one” relatingto a group or set of six elements explicitly encompasses one, two,three, four, five and six of said elements, as well as at least two, atleast three, at least four, at least five of said elements.

Each of the relevant disclosures of all references cited herein isspecifically incorporated by reference. The following examples areoffered by way of illustration, and not by way of limitation.

DEFINITIONS

The terms and names used in the present application have their ordinarymeaning in the field of HPV detection in general, and of molecularbiology in particular.

By <<amplification>>, it is meant any technique of nucleic acidamplification, such as the PCR (Polymerase Chain Reaction), or theisothermal amplification techniques, e.g., TMA (transcription mediatedamplification), NASBA (nucleic acid sequence based amplification), 3SR(self sustained sequence replication) or strand displacementamplification.

Amplification methods, especially PCR-based methods, are known in theart (Molecular Cloning: A Laboratory Manual, Maniatis, Fritsch, andSambrook, CSHL Press; Molecular Biology of the Cell, Alberts et al.; PCRPrimer: A Laboratory Manual, Dieffenbach and Dveksler, CSHL Press; ThePolymerase Chain Reaction, Mullis, Ferré, and Gibbs, Birkhauser BostonPress; Gene quantification, Ferré, Birkhauser Boston Press).

By PCR or PCR reaction, we hereby understand any PCR-based method. Thisincludes standard PCR, qualitative, quantitative and semi-quantitativePCR, real-time PCR, reverse-transcriptase PCR (RT-PCR), simplex andmultiplex PCR, and the like.

By real-time PCR, we hereby understand any PCR-based method allowing formonitoring of a signal, such as fluorescence, emitted during thereaction as an indicator of amplicon production during each PCR cycle asopposed to the endpoint detection by conventional PCR methods.

By quantitative PCR, we hereby understand any PCR-based method allowingfor the estimation of the initial amount of a given PCR target in agiven sample.

By multiplex PCR, we hereby understand any PCR reaction aiming at theamplification of more than one target. For instance, multiplex PCRinclude duplex PCR (two targets), triplex PCR (three targets), andhigher multiplex PCR. Multiplex PCR includes PCR reactions with morethan one primer pair, for instance two primer pairs. In this case, theremight be four different primers, but it is also possible for the twoprimer pairs to have one primer in common, e.g. the forward primer, andto have two distinct reverse primers. Multiplex PCR also includes PCRreactions with a unique primer pair, but with more than one probe. Theterm “megaplex” is herein sometimes used: it basically has the samemeaning as “multiplex”, but is used to distinguish multi-multiplex,which involves at least two different group-targeted systems (e.g., anA5- and an A6- and an A7- and an A9-targeted systems), from the“multiplex”, which involve one group-targeted system (e.g., an A7- or anA9-targeted system).

By nucleic acid, we hereby understand any nucleic acid: it can besynthetic or not, recombinant or naturally occurring, linear orcircular. This includes DNA and RNA. The nucleic acid can be eithersingle stranded or double stranded or even triple stranded. It can stemfrom various biological sources, such as micro organisms (bacteria,yeasts, and the like), or higher organisms, like mammal cells. Saidnucleic acid can also be of viral nature, e.g., the HPV nucleic acids.The nucleic acid can also comprise total DNA, total RNA, genomic DNA,mitochondrial DNA, plasmidic DNA, BAC DNA, and mixtures thereof.Moreover, the nucleic acid can assume various states of purity.

By oligonucleotide, we hereby understand any short polymer ofnucleotides, wherein nucleotides can be ribonucleotides,deoxyribonucleotides, dideoxyribonucleotides, degenerated nucleotides,and the like. Said oligonucleotides are preferably single-stranded. Thelength of said oligonucleotides can vary, and is usually under 150nucleotides (nt), preferably in the range of 10-100 nt, more preferably13-60 nt, even more preferably 13-50 nt. Said oligonucleotides can bearchemical modifications, such as tagging or marking, for instanceradioactive, fluorescent, biotinylated, dig labelling. Anoligonucleotide according to the invention can be either forward (sense)or reverse (antisense). In addition, it should be stressed, thatalthough preferred functions may be mentioned in relation to someoligonucleotides according to the present invention, it is obvious thata given oligonucleotide may assume several functions, and may be used indifferent ways according to the present invention. For example, anoligonucleotide can be used either as a primer, or as a probe. Also,when an oligonucleotide is described as being useful as anamplicon-targeting probe, the skilled person understands that thecomplementary sequence of this oligonucleotide is equally useful as aprobe to target the same amplicon. Moreover, it is also obvious, thatany primer suitable for a multiplex assay, can also, within the meaningof the present invention, be used in a simplex protocol. The sameapplies to a primer suitable for a real-time protocol, which can also beused in the framework of an end-point assay within the meaning of thepresent invention.

Oligonucleotides according to the invention especially include PCRprimers and probes. Unless otherwise stated, nucleic acid sequences aregiven in the 5′ to 3′ direction. Said oligonucleotides can be under manyforms, e.g. under dry state, in solution/suspension with the desiredsolvent and the desired concentration. The skilled person would know,which solvents, concentrations, storage conditions are suitable for theoligonucleotides of the invention. In particular, the skilled personwould know how to prepare said oligonucleotides as stock solutions. Theoligonucleotides according to the invention can also assume variousdegrees of purity, as can be judged by those skilled in the art, e.g.,by HPLC chromatography.

By set or systems of oligonucleotides, we hereby understand anycombination comprising at least one oligonucleotide, preferably at leasttwo, e.g. 2-10 oligonucleotides. Said set can thus comprise one PCRprimer, or a pair of PCR primers, or a probe, or a probe and a pair ofprimers. Said oligonucleotides can be separately kept, or partiallymixed, or entirely mixed.

The notion of primer or PCR primer is known to those skilled in the art.For example, it includes any oligonucleotide able to anneal to a targettemplate under suitable stringency conditions, and allowing forpolymerase strand elongation. The typical length of said primer is 13-30nt, preferably 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27nt.

The terms “primer”, “amplification primer” or “nucleic acid primer” areused interchangeably herein. A “primer” refers to a shortpolynucleotide, whether occurring naturally as in a purified restrictiondigest or produced synthetically, which is capable of acting as a pointof initiation of synthesis when placed under conditions in whichsynthesis of a primer extension product, which is complementary to anucleic acid strand, is induced, i.e., in the presence of nucleotidesand an inducing agent such as a polymerase and at a suitable temperatureand pH. The primer must be sufficiently long to prime the synthesis ofthe desired extension product. The exact length of the primer willdepend upon experimental factors, and notably upon temperature, sourceof primer and use of the process.

A primer pair consists of a forward primer and a reverse primer, whereinthe forward primer is sufficiently complementary to one HPV strand tohybridize thereto, and the reverse primer is sufficiently complementaryto the other HPV strand to hybridize thereto.

Stringency refers to hybridization conditions chosen to optimize bindingof polynucleotide sequences with different degrees of complementarity.Stringency is affected by factors such as temperature, salt conditions,the presence of organic solvents in the hybridization mixtures, and thelengths and base compositions of the sequences to be hybridized and theextent of base mismatching, and the combination of parameters is moreimportant than the absolute measure of any one factor.

Very High Stringency: Very high stringency conditions refers tohybridization to filter-bound DNA in. 5×SSC, 2% sodium dodecyl sulfate(SDS), 100 microgrammes/ml single stranded DNA at 55-65° C. for 8 hours,and washing in 0.1×SSC and 0.1% SDS at 60-65° C. for thirty minutes.

High Stringency High stringency conditions refers to hybridization tofilter-bound DNA in 5×SSC, 2% sodium dodecyl sulfate (SDS), 100microgrammes/ml single stranded DNA at 55-65° C. for 8 hours, andwashing in 0.2×SSC and 0.2% SDS at 60-65° C. for thirty minutes.

Moderate Stringency Moderate stringency conditions refers tohybridization to filter-bound DNA in 5×SSC, 2% sodium dodecyl sulfate(SDS), 100 microgrammes/ml single stranded DNA at 55-65° C. for 8 hours,and washing in 0.2×SSC and 0.2% SDS at 50-55° C. for thirty minutes.

The notion of probe is also known to those skilled in the art. Forexample, it includes any oligonucleotide able to anneal to a targettemplate under the desired hybridization conditions. The typical lengthof said probe is 15-60 nt, preferably 16-50 nt, more preferably 17-40nt, more preferably 17-35 nt, more preferably 20-30 nt. Preferably, saidprobe is fluorescently labelled. However, it is clear to those skilledin the art that under certain conditions, one may use a primer as aprobe and vice-versa. Moreover, it is herein stressed that the productsaccording to the present invention, especially, inter alia,oligonucleotides, are not limited to the intended use herein mentioned,but rather are to be broadly construed, irrespective of the indicateddestination. For instance, a claim to a product (oligonucleotide) for aparticular use should be construed as meaning a product(oligonucleotide) which is in fact suitable for the stated use. Thus, anoligonucleotide suitable for use as a primer in a multiplex protocol isalso clearly adapted to a simplex protocol within the meaning of thepresent invention.

A probe may entirely consist of a hybridizing segment. By “hybridizingsegment” or “annealing segment” of a probe, it is meant the nucleotidesequence, which is intended to anneal to the HPV target(s).

Alternatively, a probe may comprise at least one detection component,e.g. at least one detection label (such as a radioactive element, or afluorophore). This detection label can be linked to the hybridizingsegment of the probe via short HPV-unrelated oligonucleotide arms, whichare known to the skilled person as beacon arm, or Scorpion™ arm. Aprobe, which comprises at least one 5′ and/or 3′ detection label, or atleast one 5′ and/or 3′ beacon arm, consists of a hybridizing segment andof at least one 5′ and/or 3′ label or beacon arm.

Various formats (types) of probes, including Taqman™ probes (hydrolysisprobes), molecular Beacons™ (beacon probes or molecular beacon probes),and Scorpion™ probes are known in the art.

In a preferred embodiment, the probes according to the invention can allbe synthesized and used in the molecular beacon format.

The structure of molecular beacons is as follows. A short nucleotidesequence (so-called beacon arm) which is unrelated to the targetsequence is thus covalently linked to both ends of the probe. A shortunrelated arm is thus linked in 5′ of the probe, and is labelled with afluorescent moiety (i.e. fluorescent dye or fluorescent marker). Anotherbut still unrelated arm is linked to the 3′ end of probe and is labelledwith a fluorescence quenching moiety. Thus, molecular beacons have afluorophore and a quencher at opposite ends. The 5′ short arm is totallycomplementary to the one in 3′ so that they can anneal together, andthus can assume a hairpin structure when unhybridized to the target insolution. In this hairpin conformation, the quencher and the fluorescentdye are close enough to each other to allow efficient quenching of thefluorophore. However, when the probe encounters a target molecule,annealing is favoured with respect to the hairpin conformation whenvalues of beacon arm Tm and probe Tm are suitably chosen (theoretically:probe Tm>beacon arm Tm>primer Tm, wherein Tm is the melting temperatureof interest). The fluorophore and quencher move away from each other andthe fluorophore can then fluoresce when illuminated by suitable lightexcitation. As PCR proceeds, amplification product accumulates, and theamount of fluorescence at any given cycle depends on the amount ofamplification product present at that time. (See e.g., Sanjay Tyagi andFred Russell Kramer, Nature Biotechnology 1996, volume 14, pages303-308; Nature Biotechnology 1998, volume 16, pages 49-53).

(Remark: It is also possible to link the fluorophore at the 3′ end,while attaching the quencher at the 5′ end.)

Schematically, said probe can have the following formulae (molecularbeacon format):

5′Fluorophore-(arm1)-probe-(arm2)-Quencher 3′5′ Quencher-(arm1)-probe-(arm2)-Fluorophore 3′wherein arm1 and arm2 can be any short nucleotide sequences, e.g. in therange of 3-10 nucleotides, preferably 5, 6, 7 nucleotides, allowing forthe hair pin structure formation under suitable stringency conditions,i.e. arm1 and arm2 are totally complementary to anneal under the desiredstringency conditions (standard PCR stringency conditions include, forexample, an annealing temperature of 55 to 65° C. and an Mgconcentration of 4 to 8 mM). However, arm1 and arm2 are unrelated to thetarget sequence of the probe, i.e. the hairpin conformation resultingfrom the annealing between arm1 and arm2 is essentially the onlypossible secondary structure for the probe when unhybridized. Theskilled person would know how to choose such arms for a given probe.

Illustrative beacon formats include:

TGCGC-(probe sequence)-GCGCA GCGCA-(probe sequence)-TGCGC AGCGC-(probesequence)-GCGCT GCGCT-(probe sequence)-AGCGC CGCGA-(probesequence)-TCGCG CGCGC-(probe sequence)-GCGCG.

By fluorophore, it is herein understood any fluorescent marker/dye knownin the art. Examples of such suitable fluorescent markers include Fam,Hex, Tet, Joe, Rox, Tamra, Max, Edans, Cy dyes such as Cy5, Fluorescein,Coumarin, Eosine, Rhodamine, Bodipy, Alexa, Cascade Blue, Yakima Yellow,Lucifer Yellow and Texas Red (all of them are Trade-Marks), the familyof ATTO dyes.

By quencher, we herein understand any quencher known in the art.Examples of such quenchers include Dabcyl, Dark Quencher, Eclipse DarkQuencher, ElleQuencher, Tamra, BHQ and QSY (all of them areTrade-Marks).

The skilled person would know which combinations of dye/quencher aresuitable when designing a probe.

In a preferred embodiment according to the invention, spectralproperties of said probes can be chosen as to not interfere with eachother. In particular, when probes are used in multiplex, each singleprobe can have its own fluorophore being spectrally significantlydifferent from each other, i.e. the absorption/emission spectra areessentially non-overlapping. This advantageously allows for low-noisemultiplex detection for all single probes, making sure that individualsignals do not interfere with each other in detection. Examples of dyeswhich can be used together in multiplex include Fam with Tamra, Fam withTamra with Texas Red. The choice of appropriate dyes to be used togethermay also be dependent of the filter contained in the amplificationapparatus.

According to the invention, all the provided oligonucleotides can beeither kept separately, or partially mixed, or totally mixed.

Said oligonucleotides can be provided under dry form, or solubilized ina suitable solvent, as judged by the skilled person. Suitable solventsinclude TE, PCR-grade water, and the like.

The term “significantly” is herein used in its usual meaning in thefield of statistics (e.g., t test, z test, chi squared value, or Fratio, etc.), i.e., for comparing a value to another one, anddetermining whether these values differ from each other. The term“significantly” hence encompasses the fact that the skilled person maytake into account the standard deviation (if any), which measures theamount of spread of data in a frequency distribution. The desired pvalue is usually set at an alpha level of 5%, or at the more stringentalpha level of 1%.

In the examples below, are described several A6-, A5-, A9 andA7-targeted amplification and/or detection systems of the invention.

Tables 12-35: these tables give the SEQ ID NO: and positions of thereference amplicons, the forward primers, the reverse primers, theprobes, the beacons probes of illustrative group-targeted systems of theinvention.

Table 12: Reference amplicons of A5-targeted systems (from HPV51 genome)

Table 13: Forward primers of A5-targeted systems

Table 14: Reverse primers of A5-targeted systems

Table 15: Probes of A5-targeted systems

Table 16: Beacon probes of A5-targeted systems

Table 17: A5-targeted systems

Table 18: Reference amplicons of A6-targeted systems (from HPV56 genome)

Table 19: Forward primers of A6-targeted systems

Table 20: Reverse primers of A6-targeted systems

Table 21: Probes of A6-targeted systems

Table 22: Beacon probes of A6-targeted systems

Table 23: A6-targeted systems

Table 24: Reference amplicons of A7-targeted systems (from HPV18 genome)

Table 25: Forward primers of A7-targeted systems

Table 26: Reverse primers of A7-targeted systems

Table 27: Probes of A7-targeted systems

Table 28: Beacon probes of A7-targeted systems

Table 29: A7-targeted systems

Table 30: Reference amplicons of A9-targeted systems (from HPV16 genome)

Table 31: Forward primers of A9-targeted systems

Table 32: Reverse primers of A9-targeted systems

Table 33: Probes of A9-targeted systems

Table 34: Beacon probes of A9-targeted systems

Table 35: A9-targeted systems

Tables 36-50: these tables show the number of nucleotide mismatchesshown by primers and probes of the invention (alignment of the sequencesof 50 HPV types); an empty box indicates there is no coherent sequencealignment; a gray box indicates that the number of mismatch(es) is of 0,1, 2 or 3.

Table 36: mismatch numbers shown by primers and probes of A5-targetedsystems of the invention (systems A to C)

Table 37: mismatch numbers shown by primers and probes of A5-targetedsystems of the invention (systems D to E)

Table 38: mismatch numbers shown by primers and probes of A6-targetedsystems of the invention (systems A to C)

Table 39: mismatch numbers shown by primers and probes of A6-targetedsystems of the invention (systems D to E)

Table 40: mismatch numbers shown by primers and probes of A7-targetedsystems of the invention (systems A to B)

Table 41: mismatch numbers shown by primers and probes of A7-targetedsystems of the invention (systems C to D)

Table 42: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system C)

Table 43: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system E1)

Table 44: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system E2)

Table 45: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system E3)

Table 46: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system E4)

Table 47: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system F)

Table 48: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system GZ7)

Table 49: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system GZ8)

Table 50: mismatch numbers shown by primers and probes of A9-targetedsystems of the invention (system H)

Tables 51-68: Specificity of the Detection Systems of the Invention

The amplification systems described in tables 36-50 have been used totest the specificity of the probes of the invention.

Table 51: illustrative list of HPV plasmids, which can be used to testthe HPV specificity of the detection systems of the invention; the wholelist of plasmids have been used for the specificity results, which areherein described

Table 52: illustrative PCR material and method conditions, which can beused to test the specificity of the A5- and A6-targeted detectionsystems of the invention

Table 53: specificity results of A5-targeted probes (box “other HPV”=allthe other HPV of the list given in Table 51)

Table 54: specificity results of A6-targeted probes (box “other HPV”=allthe other HPV of the list given in Table 51)

Table 55: illustrative PCR material and method conditions, which can beused to test the specificity of the A7-targeted detection systems of theinvention

Tables 56-59: specificity results of A7-targeted probes (box “otherHPV”=all the other HPV of the list given in Table 51)

-   -   Table 56: A7-targeted amplification system A, and probes of        A7-targeted amplification system A (one probe per PCR)    -   Table 57: A7-targeted amplification system B, and probes of        A7-targeted amplification system B (one probe per PCR)    -   Table 58: A7-targeted amplification system C, and probes of        A7-targeted amplification system C (one probe per PCR)    -   Table 59: A7-targeted amplification system D, and probes of        A7-targeted amplification system D (one probe per PCR)

Table 60: illustrative PCR material and method conditions, which can beused to test the specificity of the A9-targeted detection systems of theinvention

Tables 61-68: specificity results of A9-targeted probes (box “otherHPV”=all the other HPV of the list given in Table 51)

-   -   Table 61: A9-targeted amplification system C, and probes of        A9-targeted amplification system C (one probe per PCR)    -   Table 62: A9-targeted amplification system E2, and probes of        A9-targeted amplification system E2 (one probe per PCR)    -   Table 63: A9-targeted amplification system E3, and probes of        A9-targeted amplification system E3 (one probe per PCR)    -   Table 64: A9-targeted amplification system E4, and probes of        A9-targeted amplification system E4 (one probe per PCR)    -   Table 65: A9-targeted amplification system F, and probes of        A9-targeted amplification system F (one probe per PCR)    -   Table 66: A9-targeted amplification system GZ7, and probes of        A9-targeted amplification system GZ7 (one probe per PCR)    -   Table 67: A9-targeted amplification system GZ8, and probes of        A9-targeted amplification system GZ8 (one probe per PCR)    -   Table 68: A9-targeted amplification system H, and probes of        A9-targeted amplification system H (one probe per PCR)

The amplification and detection systems, which were used for thespecificity tests, are those shown in Table 17 (A5-targeted systems);Table 23 (A6-targeted systems); Table 29 (A7-targeted systems); Table 35(A9-targeted systems).

For the A9-targeted systems, those primers, which are shown betweenbrackets in Table 35 were not used, as they would have been redundant:indeed, those primers, which are not between brackets, are sufficient toamplify all group A9 HPV. Hence, in table 35, those A9-targeted primerswhich are shown between brackets are optional and/or equivalent and/oralternative primers, if it is wished to amplify all group A9 HPV.

Tables 69-82: System Sensitivity

The sensitivity of systems of the invention has been tested under thesame experimental conditions, as for the specificity tests (see Tables52, 55, 60).

Table 69: sensitivity of A5-targeted systems (systems A, B, C, D and E)

Table 70: sensitivity of A6-targeted systems (systems A, B, C, D and E)

Tables 71-74: Sensitivity of A7-Targeted Systems

-   -   Table 71: sensitivity of A7-targeted system A    -   Table 72: sensitivity of A7-targeted system B    -   Table 73: sensitivity of A7-targeted system C    -   Table 74: sensitivity of A7-targeted system D

Tables 75-82: Sensitivity of A9-Targeted Systems

-   -   Table 75: sensitivity of A9-targeted system C    -   Table 76: sensitivity of A9-targeted system E2    -   Table 77: sensitivity of A9-targeted system E3    -   Table 78: sensitivity of A9-targeted system E4    -   Table 79: sensitivity of A9-targeted system F    -   Table 80: sensitivity of A9-targeted system GZ7    -   Table 81: sensitivity of A9-targeted system GZ8    -   Table 82: sensitivity of A9-targeted system H

Tables 83-88: “Megaplex” Specificity and Sensitivity

PCR runs have been conducted with one A5-targeted system, oneA6-targeted system, one A7-targeted system, one A9-targeted system, in asingle-tube amplification.

As the A7- and A9-targeted systems already are multiplex systems (i.e.,they each have more than two primers), the mix of the fourgroup-targeted systems is herein referred to as a “megaplex”.

The megaplex PCR have been tested with the plasmids listed in Table 51,in specificity (Ct; RFU) and in sensitivity (Ct; RFU).

Tables 83-86: Specificity Results for Megaplex EAAH and EBAC

-   -   Tables 83-84: specificity of the megaplex EAAH    -   Table 83: illustrative megaplex material and method conditions,        which can be used for a mix of A5-targeted system E, A6-targeted        system A, A7-targeted system A and A9-targeted system H (i.e.,        megaplex EAAH); these experimental conditions have been used for        the results depicted in table 84    -   Table 84: specificity results of the EAAH megaplex    -   Tables 85-86: specificity of the megaplex EBAC    -   Table 85: illustrative megaplex material and method conditions,        which can be used for a mix of A5-targeted system E, A6-targeted        system B, A7-targeted system A and A9-targeted system C (i.e.,        megaplex EBAC); these experimental conditions have been used for        the results depicted in table 86;    -   Table 86: specificity results of the EBAC megaplex

Tables 87-88: Sensitivity Results of the Megaplex EAAH and EBAC

-   -   Table 87: sensitivity results of the megaplex EAAH    -   Table 88: sensitivity results of the megaplex EBAC

Table 89: list of HPV genome sequences.

These tables are followed by a listing of sequences of referencetemplates.

A5 Group=HPV51; HPV26; HPV69; HPV82 A5 HR HPV=HPV51 A5 ReferenceGenome=HPV51 (NC_(—)001533.1; Human HPV, Complete Genome)

TABLE 12 A5 REFERENCE AMPLICON SEQUENCES (from reference genome) =

TABLE 13 A5 FORWARD PRIMERS Size SEQ ID Name Sequence Address (bp) NO:A5E6f1 GGCAGTGGAAAGCAGTGGAGAC 772 22 6 A5E6f2 AGCTCCGTGTTGCAGGTGTTC 73621 7 A5E6f3 ATATGCGTGACCAGCTACCAG 678 21 8 A5E6f4 GACAGGCTACGTGTTACAGAA711 21 9 A5E6f5 CGGGCTGGACAGGCTACG 704 18 10

TABLE 14 A5 REVERSE PRIMERS Size SEQ ID Name Sequence Address bp NO:A5E6r1 CCATCGCCGTTGCTAGTTGTTC 877 22 11 A5E6r2 AGTCCATCGCCGTTGCTAGTTG880 22 12 A5E6r3 TGTCTCCACTGCTTTCCACTG 794 21 13 A5E6r4CCCTCATCCTCTGTACCTTC 902 20 14 A5E6r5 TCGCCCATTAACATCTGCTGT 828 21 15

TABLE 15 A5 PROBES Corresponding Size beacon probes Sequence Address(bp) SEQ ID NO: A5E6s1, A5E6s1b GCTTAGTTCGCCCATTAACATCTGCTG 835 27 16A5E6s2 CGAAGGGTGTCTCCACTGCTTTCCA 801 25 17 A5E6s3ACACGGAGCTTCAATTCTGTAACACG 745 26 18 A5E6s4 TAGTACAACTGGCAGTGGAAAGCAGT762 26 19

TABLE 16 A5 BEACON PROBES Name Sequence (underlined are shown the beaconarms) Address SEQ ID NO: A5E6s1CCCCCTCGCTTAGTTCGCCCATTAACATCTGCTGGAGGGGG 835 20 A5E6s1bCGCTGCGCTTAGTTCGCCCATTAACATCTGCTGGCAGCG 835 21 A5E6s2CGCGATCCGAAGGGTGTCTCCACTGCTTTCCAGATCGCG 801 22 A5E6s3CGCGATCACACGGAGCTTCAATTCTGTAACACGGATCGCG 745 23 A5E6s4CGCGATCTAGTACAACTGGCAGTGGAAAGCAGTGATCGCG 762 24 A5E6s4CGCGATCTAGTACAACTGGCAGTGGAAAGCAGTGATCGCG 762 24

TABLE 17 A5 systems; minimal set = one forward primer, one reverseprimer, and one probe

A6 Group=HPV30; HPV53; HPV56; HPV66 A6 HR HPV=HPV56 A6 ReferenceGenome=HPV56 (NC_(—)001594.1; Human HPV56, Complete Genome)

TABLE 18 A6 REFERENCE AMPLICON SEQUENCES (from reference genome) =

TABLE 19 A6 FORWARD PRIMERS Size SEQ Name Sequence Address (bp) ID NO:A6E6f1 TGGACCGGGTCATGTTTGGG 504 20 30 A6E6f2 CTAATAGCACATGGTTGGACCG 48922 31 A6E6f3 AAGGTGCTACAGATGTCAAAG 413 21 32 A6E6f4GTTGGACCGGGTCATGTTTGG 502 21 33 A6E6f5 TCAGAGGATGAGGATGAGGATG 665 22 34

TABLE 20 A6 REVERSE PRIMERS Size SEQ Name Sequence Address bp ID NO:A6E6r1 ACGTCTTGCAGCGTTGGTAC 603 20 35 A6E6r1 ACGTCTTGCAGCGTTGGTAC 603 2035 A6E6r2 GGTTCTCTAGATGTTTGTCTCC 550 22 36 A6E6r1 ACGTCTTGCAGCGTTGGTAC603 20 35 A6E6r3 ACTGCACCACAAACTTACACTC 791 22 37

TABLE 21 A6 PROBES Corresponding beacon probes Sequence Address Size(bp) SEQ ID NO: A6E6s1 ACATCTAGAGAACCTAGAGAATCTACAGTA 537 30 38 A6E6s2,A6E6s2b GGTCCAACCATGTGCTATTAGATGAA 509 26 39 A6E6s3, A6E6s3bCGGCCACAGCAAGCTAGACA 707 20 40

TABLE 22 A6 BEACON PROBES Name Sequence (underlined are shown the beaconarms) Address SEQ ID NO: A6E6s1CGCGATCACATCTAGAGAACCTAGAGAATCTACAGTAGATCGCG 537 41 A6E6s1CGCGATCACATCTAGAGAACCTAGAGAATCTACAGTAGATCGCG 537 41 A6E6s2CGCGATCGGTCCAACCATGTGCTATTAGATGAAGATCGCG 509 42 A6E6s2bCGCCTCGGTCCAACCATGTGCTATTAGATGAAGAGGCG 509 43 A6E6s1CGCGATCACATCTAGAGAACCTAGAGAATCTACAGTAGATCGCG 537 41 A6E6s3CGCGACGGCCACAGCAAGCTAGACATCGCG 707 44 A6E6s3bCGCCTCCGGCCACAGCAAGCTAGACAGAGGCG 707 45

TABLE 23 A6 SYSTEMS; minimal set = one forward primer, one reverseprimer, and one probe

A7 Group=HPV18; HPV39; HPV45; HPV59; HPV68; HPV70; HPV85 A7 HRHPV=HPV18; HPV39; HPV45; HPV59; HPV68 A7 Reference Genome=HPV18(NC_(—)001357.1; Human HPV, Complete Genome)

TABLE 24 A7 REFERENCE AMPLICON SEQUENCES (from reference genome) =

TABLE 25 A7 FORWARD PRIMERS Name Sequence Address Size (bp) SEQ ID NO:A7E16f1a TGGTATAGAACAGGAATATCAAAT 1895 24 68 A7E16f2aGAACAGGTATATCCAATATTAGTG 1902 24 69 A7E16f3a GAACAGGAATGTCCAATATTAG 190222 70 A7E115f1a TGGTATAGAACAGGAATATCAAATAT 1895 26 71 A7E115f2aGTACAGAACAGGAATGTCCAA 1897 21 72 A7E115f3d GGTATCGCACAGGTATATCC 1896 2073 A7E17f1 TGATAGCAATTTTGATTTGTCAG 1987 23 74 A7E17f2GATAGCGTATTTGACCTATCAG 1988 22 75 A7E17f3 GGAATAGATGATAGTGTATTTGATC 197925 76 A7E12f1 GGCCGATCCAGAAGGTACAGAC  916 22 77 A7E12f2CAATCGTGAAGGTACAGATGG  920 21 78

TABLE 26 A7 REVERSE PRIMERS Name Sequence Address Size bp SEQ ID NO:A7E16r1b CATTGCTGTTGCAGTCTG 2099 18 79 A7E16r2b GCAGCATTACTGTTACAATC2103 20 80 A7E16r3b CGGCGTTACTATTACTATCTG 2102 21 81 A7E115r1aTGCCATATCGCTTTCATCTG 2062 20 82 A7E115r2b AAATGCTATATCACTTTCATCTG 206523 83 A7E17r1 GCATTACTGTTGCTGTCTG 2100 19 84 A7E17r2GCGGCATTACTATTACAATCTG 2103 22 85 A7E12r2 GCATTTTCATCCTCATCCTCTG 1032 2286 A7E12r3 CCTGTGTCTGTTGCATTTTC 1044 20 87

TABLE 27 A7 PROBES Size Corresponding Sequence Address (bp) SEQ ID NO:beacon probes CAGATGAAAGCGATATGGCATT 2043 22 88 A7E1ZAS61fCAGATGAAAGTGATATTGCATAT 2043 23 89 A7E1ZAS63f CTGATGAAAGTGACATAGCATTT2043 23 90 A7E1ZAS64f CAGATGAAAGTGATATGGCATTT 2043 23 91 A7E1ZCS40fTGGAATAGATGATAGTGTATTTGAT 1978 25 92 A7E1ZBS74f GATAGCAATTTTGATTTGTCAGA1988 23 93 A7E1ZBS26f TGGAATAGATGATAGTGTATTTGAT 1978 25 92 A7E1ZBS74fAGTTGATGATAGCGTGTTTGAC 1981 22 94 A7E1ZBS79f AGTTGATGATAGCGTGTTTGAC 198122 94 A7E1ZBS80f CGATAGTAATTTTGATTTGTCAGA 1987 24 95 A7E1ZBS27fCAGATGAAAGTGATATGGCATTT 2043 23 91 A7E1ZCS11f CAGATGAAAGTGATATGGCATTT2043 23 91 A7E1ZCS40f CTGATGAAAGTGACATAGCATTT 2043 23 90 A7E1ZCS45fCAGATGAAAGTGATATTGCATAT 2043 23 89 A7E1ZCS63f AATGAGTTAACAGATGAAAGTGA2032 23 96 A7E1ZCS90f GTAATGGCTGGTTCTTTGTAGAAACAA  954 27 97 A7E1ZDS36fGTAACGGCTGGTTTTATGTACAAGCTA  954 27 98 A7E1ZDS37fGTAATGGATGGTTTTTTGTACAGGCAAT  954 28 99 A7E1ZDS38fGTAACGGATGGTTTTTTGTACAAGCAAT  954 28 100  A7E1ZDS2fGGTGTAATGGCTGGTTCTTTGTAGA  951 25 101  A7E1ZDS3fGGTGTAATGGCTGGTTCTTTGTAGA  951 25 101  A7E1ZDS4fGGTGTAATGGCTGGTTCTTTGTAGA  951 25 101  A7E1ZDS11f

TABLE 28 A7 BEACON PROBES Name Sequence (underlined are shown the beaconarms) Address SEQ ID NO: A7E1ZAS61f CGACGTCAGATGAAAGCGATATGGCATTACGTCG2043 102 A7E1ZAS63f CGACGTCAGATGAAAGTGATATTGCATATACGTCG 2043 103A7E1ZAS64f CGACGTCTGATGAAAGTGACATAGCATTTACGTCG 2043 104 A7E1ZCS40fCCGAGTCAGATGAAAGTGATATGGCATTTACTCGG 2043 105 A7E1ZBS74fACGTCGTGGAATAGATGATAGTGTATTTGATCGACGT 1978 106 A7E1ZBS26fCGCAGTGATAGCAATTTTGATTTGTCAGAACTGCG 1988 107 A7E1ZBS74fACGTCGTGGAATAGATGATAGTGTATTTGATCGACGT 1978 106 A7E1ZBS79fACGTCGAGTTGATGATAGCGTGTTTGACCGACGT 1981 108 A7E1ZBS80fCCGGCTAGTTGATGATAGCGTGTTTGACAGCCGG 1981 109 A7E1ZBS27fCGCAGTCGATAGTAATTTTGATTTGTCAGAACTGCG 1987 110 A7E1ZCS11fCGCAGTCAGATGAAAGTGATATGGCATTTACTGCG 2043 111 A7E1ZCS40fCCGAGTCAGATGAAAGTGATATGGCATTTACTCGG 2043 105 A7E1ZCS45fCGTCGTCTGATGAAAGTGACATAGCATTTACGACG 2043 112 A7E1ZCS63fCGAGGTCAGATGAAAGTGATATTGCATATACCTCG 2043 113 A7E1ZCS90fCCACGTAATGAGTTAACAGATGAAAGTGAACGTGG 2032 114 A7E1ZDS36fCGCGACGTAATGGCTGGTTCTTTGTAGAAACAAGTCGCG  954 115 A7E1ZDS37fCGCGATCGTAACGGCTGGTTTTATGTACAAGCTAGATCGCG  954 116 A7E1ZDS38fCGCGATCGTAATGGATGGTTTTTTGTACAGGCAATGATCGCG  954 117 A7E1ZDS2fCGCGCTGTAACGGATGGTTTTTTGTACAAGCAATAGCGCG  954 118 A7E1ZDS3fCGCGATGGTGTAATGGCTGGTTCTTTGTAGAATCGCG  951 119 A7E1ZDS4fCGCGATGGTGTAATGGCTGGTTCTTTGTAGAGATCGCG  951 120 A7E1ZDS11fCTCGCTCGGTGTAATGGCTGGTTCTTTGTAGAGAGCGAG  951 121

TABLE 29 A7 SYSTEMS

A9 Group=HPV16; HPV31; HPV33; HPV35; HPV52; HPV58; HPV67 A9 HRHPV=HPV16; HPV31; HPV33; HPV35; HPV52; HPV58 A9 Reference Genome=HPV16(NC_(—)001526.1; Human HPV16, Complete Genome)

TABLE 30 A9 REFERENCE AMPLICON SEQUENCES (from reference genome) =

TABLE 31 A9 FORWARD PRIMERS Forward Primer Name 5′ Sequence 3′ addresssize bp SEQ ID NO: A9E1f7 AGGACGTGGTCCAGATTAAGTTT 2707 23 211 A9E1f8AGGACGTGGTGCAGATTAAG 2707 20 212 A9E1f9 AGGACGTGGTGCAAATTAAGTTT 2707 23213 A9E1f10 AGGACGTGGTGCAGATTAAATTT 2707 23 214 A9E1f11AGGACGTGGTGCAGATTAGGTTT 2707 23 215 A9E1f12 AGGACGTGGTGCAAATTAAATTT 270723 216 A9E1f13 AGGACGTGGTGCAAATTAGGTTT 2707 23 217 A9E2f1TAGTAACACTACACCCATAGTACAT 3600 25 218 A9E2f2 TCTAACGTTGCACCTATCGTG 360021 219 A9E2f4 TCCTTCTACTGCACCTATAATACA 3600 24 220 A9E2f1aTAGTACCACTACACCCATAGTACAT 3600 25 221 A9E2f2a TCTAACGTTGCACCTATCGTGCAT3601 24 222 A9E2f4a TCCTTCTACTGCACCTATAATACAC 3600 25 223 A9E2Z5Z6f1cACTACACCTATAGTACATTTAAAAGG 3607 26 224 A9E2Z5Z6f2cGCACCTATAGTGCATTTAAAAG 3610 22 225 A9E2Z5Z6f3b TGCACCTATAATACACCTAAAAG3609 23 226 A9E21f1az TAAAAGGTGATGCTAATACTTTAAA 3626 25 227 A9E21f2bzTAAAAGGTGATGCAAATACATTAAA 3626 25 228 A9E21f3dz GCATTTAAAAGGTGAATCAAATAG3621 24 229 A9E21f4cz CTAAAAGGTGATCCTAATAGTTTAAA 3625 26 230 A9E2f5GTCGTCTACATGGCATTGGA 3699 20 231 A9E2f6 CAAGATGCTTCATCTACATGGAG 3691 23232 A9E2f7 AGAAGCGTCATCTACATGGAG 3693 21 233 A9E2f8AGTGTCGTCTACATGGCATTG 3696 21 234 A9E2f9 ATATGTCATCTACATGGCATTGG 3695 23235 A9E2f10 TGTCATCCACATGGCATTGG 3698 20 236 A9E2f10bATGTCATCCACATGGCATTG 3697 20 237 A9E2f11 TTCATCTACCTGGAGTTGGAC 3699 21238 A9E2f12 TTTCATCTACATGGAGTTGGAC 3698 22 239

TABLE 32 A9 REVERSE PRIMERS Reverse Primer Name 5′ Sequence 3′ addresssize bp SEQ ID NO: A9E1r5 TGTCCTGACACACATTTAAACG 2794 22 240 A9E1r6TGTCCTGCACTGCATTTAAAC 2794 21 241 A9E2r1 ATTGGTCACGTTGCCATTC 3790 19 242A9E2r2 AAAATTGTTGACGTTGTGTTTC 3793 22 243 A9E2r3 AACTGTTGACGTTGTGTTTC3791 20 244 A9E2r4 ACATTTGTCGTTGCGGTTC 3790 19 245 A9E2r13GTCTCTTTGTGATGTACTTATATATG 3797 26 246 A9E2r14 CCCTTTGATATTCTGTTGTGTAAG3795 24 247 A9E21r1cz TGGTCACGTTGCCATTC 3788 17 248 A9E21r2azAAAATCGTCTCTTTGTGATGT 3792 21 249 A9E21r3az AAACATTTGTTGTTGCTGTTC 379221 250 A9E21r4fz ATTTATCCCTTTGATATTCTGTTG 3790 24 251 A9E21r5azAAACAGTTGACGTTGTGTTTC 3792 21 252 A9E21r6az AAACTGTTGACGTTGTGTTTC 379221 253 A9E21r7az AAATTGTTGACGTTGTGTTTC 3792 21 254 A9E21r8azACAGTTGTCGTTGTGTTTC 3790 19 255 A9E2r7C AAATCCTGTAGACACTGTAACAGT 3840 24256 A9E2r8 ACTTATTTGCACAGTAGGTGGT 3831 22 257 A9E2r10CTTACTTGCACAGTAGTTGGTA 3830 22 258 A9E2r12 ATCCTGTTGACACTGATACTGT 383722 259 A9E2r12B TATCCTGTAGACACTGAAACTGTG 3840 24 260 A9E2r15AAATCCAGTAGACACTGTAATAGTT 3840 25 261 A9E2r7B ATCCTGTAGACACTGTAACAGTT3838 23 262 A9E2r8 ACTTATTTGCACAGTAGGTGGT 3831 22 257 A9E2r9CTTACTTGCACAGTAGGTGGTA 3830 22 263 A9E2r10 CTTACTTGCACAGTAGTTGGTA 383022 258 A9E2r12 ATCCTGTTGACACTGATACTGT 3838 22 259 A9E2r7CAAATCCTGTAGACACTGTAACAGT 3840 24 256 A9E2r12B TATCCTGTAGACACTGAAACTGTG3839 24 264 A9E2r15 AAATCCAGTAGACACTGTAATAGTTT 3838 26 261 A9E2r16ACCGTACTTATTTGCACAGTG 3837 21 265

TABLE 33 A9 PROBES 5′ Sequence 3′ address size pb SEQ ID NO:Corresponding beacon probes TCCATCGTTTTCCTTGTCCTCT 2738 22 266 A9E1S10and S10a TCCATCGTTTTCTTTGACCTCT 2738 22 267 A9E1S11 and S11aTCCATCATTTTCTTTGACCTCT 2738 22 268 A9E1S12, S12a and S12bTCTCCATCATTTTCTTTGTCCTCT 2738 24 269 A9E1S13a, S13b and S13cCTCCATCGTTTTCTTTGTCCTC 2739 22 270 A9E1S14a CTCCATCATTTTCTTTGACCTCTC2737 24 271 A9E1S15a and 15b AGTGTCGTCTACATGGCATTGGAC 3696 24 272A9E2Z7S1 ATATGTCATCCACCTGGCATTGGAC 3695 25 273 A9E2Z7S2 and S2aATATGTCATCCACCTGGCATTGGA 3695 24 274 A9E2Z7S2b ATGCTTCATCTACATGGAGATGGAC3695 25 275 A9E2Z7S3 and S3a CAAGTTTCATCTACATGGCATTGGAC 3694 26 276A9E2Z7S4 and S4a GATAGTGAATGGCAACGTGA 3766 20 277 A9E2Z8S2f, S21f andS28f ATAAGTACATCACAAAGAGACGA 3766 23 278 A9E2Z8S56f, S58f and S61fTAACTGAACAGCAACAACAAATG 3767 23 279 A9E2Z8S101f, 105f and 127fCACAACAGAATATCAAAGGGATAAATT 3765 27 280 A9E2Z8S146f, 155f and 156fCGTACAGTGATGAAACACAAC 3761 21 281 A9E2Z8S210f AACGGAAACACAACGACAAC 376820 282 A9E2Z8S231f, 236f and 250f

TABLE 34 A9 BEACON PROBES Beacon probe Name 5′ Sequence 3′ address sizebp SEQ ID NO: A9E1S10 CGCGATTCCATCGTTTTCCTTGTCCTCTATCGCG 2738 22 283A9E1S10a CGCGATCCATCGTTTTCCTTGTCCTCTTCGCG 2738 22 284 A9E1S11CGCGATTCCATCGTTTTCTTTGACCTCTATCGCG 2738 22 285 A9E1S11aCGCGATCCATCGTTTTCTTTGACCTCTTCGCG 2738 22 286 A9E1S12CGCGATTCCATCATTTTCTTTGACCTCTATCGCG 2738 22 287 A9E1S12aCGCGATCCATCATTTTCTTTGACCTCTTCGCG 2738 22 288 A9E1S12bCGCTGTCCATCATTTTCTTTGACCTCTCAGCG 2738 22 289 A9E1S13aCGCGTTCTCCATCATTTTCTTTGTCCTCTACGCG 2738 24 290 A9E1S13bCGCCGTCTCCATCATTTTCTTTGTCCTCTCGGCG 2738 24 291 A9E1S13cCGCGATTCTCCATCATTTTCTTTGTCCTCTATCGCG 2738 24 292 A9E1S14aCGCGATCTCCATCGTTTTCTTTGTCCTCATCGCG 2739 22 293 A9E1S15aCGCCGCTCCATCATTTTCTTTGACCTCTCCGGCG 2737 24 294 A9E1S15bCGCGATCTCCATCATTTTCTTTGACCTCTCATCGCG 2737 24 295 A9E2Z7S1CGCGAAGTGTCGTCTACATGGCATTGGACTCGCG 3696 24 296 A9E2Z7S2CGCTCGATATGTCATCCACCTGGCATTGGACCGAGCG 3695 25 297 A9E2Z7S2aCGCATGATATGTCATCCACCTGGCATTGGACCATGCG 3695 25 298 A9E2Z7S2bCGCATGATATGTCATCCACCTGGCATTGGACATGCG 3695 24 299 A9E2Z7S3CGCACTATGCTTCATCTACATGGAGATGGACAGTGCG 3695 25 300 A9E2Z7S3aCCGACGATGCTTCATCTACATGGAGATGGACCGTCGG 3695 25 301 A9E2Z7S4CGCGATCAAGTTTCATCTACATGGCATTGGACATCGCG 3694 26 302 A9E2Z7S4aCGCGAGCAAGTTTCATCTACATGGCATTGGACCTCGCG 3694 26 303 A9E2Z8S2fCAGCGTGATAGTGAATGGCAACGTGAACGCTG 3766 20 304 A9E2Z8S21fCGGACTGATAGTGAATGGCAACGTGAAGTCCG 3766 20 305 A9E2Z8S28fCTCGCTGATAGTGAATGGCAACGTGAAGCGAG 3766 20 306 A9E2Z8S56fCGAGCTATAAGTACATCACAAAGAGACGAAGCTCG 3766 23 307 A9E2Z8S58fCGCAGTATAAGTACATCACAAAGAGACGAACTGCG 3766 23 308 A9E2Z8S61fCGCGTTATAAGTACATCACAAAGAGACGAAACGCG 3766 23 309 A9E2Z8S101fCGAGGTTAACTGAACAGCAACAACAAATGACCTCG 3767 23 310 A9E2Z8S105fCGCGATTAACTGAACAGCAACAACAAATGATCGCG 3767 23 311 A9E2Z8S127fCCGGCTTAACTGAACAGCAACAACAAATGAGCCGG 3767 23 312 A9E2Z8S146fCGCGATCACAACAGAATATCAAAGGGATAAATTATCGCG 3765 27 313 A9E2Z8S155fCGCACGCACAACAGAATATCAAAGGGATAAATTCGTGCG 3765 27 314 A9E2Z8S156fCCGGCTCACAACAGAATATCAAAGGGATAAATTAGCCGG 3765 27 315 A9E2Z8S210fCCGGCTCGTACAGTGATGAAACACAACAGCCGG 3761 21 316 A9E2Z8S231fCGAGGTAACGGAAACACAACGACAACACCTCG 3768 20 317 A9E2Z8S236fCGCGTTAACGGAAACACAACGACAACAACGCG 3768 20 318 A9E2Z8S250fCGATGCAACGGAAACACAACGACAACGCATCG 3768 20 319

TABLE 35 A9 SYSTEMS

Those primers which are between brackets are optional and/or equivalentand/or alternative primers.

TABLE 36 A5 Systems A to C: sequence aligment mismach evaluationReference of sequence: HPV 56->ref|NC_001594.1|

TABLE 37 A5 Systems D & E: sequence aligment mismach evaluationReference of sequence: HPV 56->ref|NC_001594.1|

TABLE 38 A6 Systems A to C: sequence aligment mismach evaluationReference of sequence: HPV 56->ref|NC_001594.1|

TABLE 39 A6 Systems D & E: sequence aligment mismach evaluationReference of sequence: HPV 56->ref|NC_001594.1|

TABLE 40 A7 System A & B: sequence aligment mismach evaluation Referenceof sequence: HPV 18->gi|9626069|ref|NC_001357.1|

TABLE 41 A7 System C & D: sequence aligment mismach evaluation Referenceof sequence: HPV 18->gi|9626069|ref|NC_001357.1|

TABLE 42 A9 System C: sequence aligment mismach evaluation Reference ofsequence: HPV 16->gi|9627100|ref|NC_001526.1|

TABLE 43 A9 System E1: sequence aligment mismach evaluation Reference ofsequence: HPV 16->gi||ref|NC_001526.1|

TABLE 44 A9 System E2: sequence aligment mismach evaluation Reference ofsequence: HPV 16->gi||ref|NC_001526.1|

TABLE 45 A9 System E3: sequence aligment mismach evaluation Reference ofsequence: HPV 16->gi||ref|NC_001526.1|

TABLE 46 A9 System E4: sequence aligment mismach evaluation

TABLE 47 A9 System F: sequence aligment mismach evaluation

TABLE 48 A9 System GZ7: sequence aligment mismach evaluation

TABLE 49 A9 System GZ8: sequence aligment mismach evaluation

TABLE 50 A9 System H

TABLE 51 list of HPV plasmids Name Group Plasmid size kb Insert size kbSource Publications pHPV 16 A9 2.961 7.904 ATCC 45113 pHPV 6B A10 2.6867.900 ATCC 45150 The EMBO Journal vol2 n^(o)12 p.2341-2348 (1983) pHPV18 A7 4.363 7.857 ATCC 45152 J. Mol. Biol. (1987) 193 p.599-608 pHPV 31A9 4.363 8.000 ATCC 65446 J Virol 58: 225-229, 1986 pHPV 11 A10 4.3637.931 ATCC 45151 Virology 151 124-130 (1986) pHPV 35 cl 2A A9 4.3633.750 ATCC 40330 U.S. Pat. 4,849,332 pHPV 35 cl 2B A9 4.363 4.100 ATCC40331 U.S. Pat. 4,849,332 pHPV 56 cl 2A A6 2.818 5.100 ATCC 40341 U.S.Pat. 4,908,306 pHPV 56 cl 2C A6 2.818 7.900 ATCC 40549 U.S. Pat.4,908,306 pHPV 56 cl 2B A6 2.818 3.100 ATCC 40379 U.S. Pat. 4,908,306pHPV 43 cl 2A A8 2.812 6.300 ATCC 40338 U.S. Pat. 4,849,334 pHPV 43 cl2B A8 2.812 2.850 ATCC 40339 U.S. Pat. 4,849,334 pHPV 44 cl 2 A10 2.8187.800 ATCC 40353 U.S. Pat. 4,849,331 pHPV 7cl 7/4 A8 2.686 3.905 DKFZpHPV 7cl 7/5 A8 2.686 4.131 DKFZ pHPV 13 cl 13 A10 2.686 7.241 DKFZ pHPV30 cl 30 A6 4.36 7.157 DKFZ pHPV 40 cl 40 A8 2.686 7.296 DKFZ pHPV 53 cl53 A8 3.939 7.154 DKFZ pHPV 57 cl 57 A4 2.686 7.235 DKFZ pHPV 72 cl 72A3 2.961 7.307 DKFZ pHPV 73 cl 73 A11 2.961 7.005 DKFZ pHPV 45 cl 45 A72.871 7.149 DKFZ pHPV 51 A5 2.68 7.800 DKFZ J. of Virology 1998p1452-1455/aug.1991 p.4216-4225 pHPV 26 A5 2.686 7.100 DKFZ pHPV 52 A92.686 7.940 DKFZ pHPV 89 Frag 1 A3 3.015 0.700 DKFZ The Journal ofinfectious diseases 2002 185: 1794-7 pHPV 89 Frag 2 A3 3.015 1.100 DKFZThe Journal of infectious diseases 2002 185: 1794-7 pHPV 89 Frag 3 A33.015 2000 DKFZ The Journal of infectious diseases 2002 185: 1794-7 pHPV89 Frag 4 A3 3.015 5.100 DKFZ The Journal of infectious diseases 2002185: 1794-7 pHPV 62 Frag 1 A3 3.015 3.325 DKFZ pHPV 62 Frag 2 A3 3.0154.040 DKFZ pHPV 62 Frag 3 A3 3.015 1.268 DKFZ pHPV 84 Frag 1 A3 3.0150.700 DKFZ Virology 279, 109-115, 2001 pHPV 84 Frag 2 A3 3.015 4.500DKFZ Virology 279, 109-115, 2001 pHPV 84 Frag 3 A3 3.015 1000 DKFZVirology 279, 109-115, 2001 pHPV 84 Frag 4 A3 3.015 1.800 DKFZ Virology279, 109-115, 2001 pHPV 84 Frag 5 A3 3.015 0.600 DKFZ Virology 279,109-115, 2001 pHPV 90 Frag 1 A3 3.015 4.200 DKFZ The Journal ofinfectious diseases 2002 185: 1794-7 pHPV 90 Frag 2 A3 3.015 1.700 DKFZThe Journal of infectious diseases 2002 185: 1794-7 pHPV 90 Frag 3 A33.015 2.500 DKFZ The Journal of infectious diseases 2002 185: 1794-7pHPV 86 Frag 1 A3 3.015 3.900 DKFZ J Gen Virol 2001, 82, 2035-2040 pHPV86 Frag 2 A3 3.015 5.800 DKFZ J Gen Virol 2001, 82, 2035-2040 pHPV 86Frag 3 A3 3.015 0.140 DKFZ J Gen Virol 2001, 82, 2035-2040 pHPV 91 Frag1 A8 3.015 3.200 DKFZ The Journal of infectious diseases 2002 185:1794-7 pHPV 91 Frag 2 A8 3.015 1.500 DKFZ The Journal of infectiousdiseases 2002 185: 1794-7 pHPV 91 Frag 3 A8 3.015 1.400 DKFZ The Journalof infectious diseases 2002 185: 1794-7 pHPV 91 Frag 4 A8 3.015 2.500DKFZ The Journal of infectious diseases 2002 185: 1794-7 pHPV 33 A94.363 7.093 CNCM I-450 pHPV 39 A7 3.005 7.160 CNCM I-507 JCM mar 1996,738-744 pHPV 42 A1 3.030 7.107 CNCM I-508 pHPV 54 A4 3.030 7.107 CNCMI-756 pHPV 23 B1 4.363 7.324 CNCM I-391 J Virol, december 1984, 52,1013-1018 pHPV 68 A7 4.363 6.042 CNCM I-1540 JCM march 1996,738-744/U.S. Pat. 5,981,173 pHPV 66 A6 4.363 7.158 CNCM I-951 J virol,1986, 57, 688-692 pHPV 87 L1 E1 16 A3 3.015 1.014 DKFZ Journal ofVirology déc. 2001 p11913-11919 pHPV 87 L1 f A3 3.015 0.974 DKFZ Journalof Virology déc. 2001 p11913-11919 pHPV 87 MY 16 A3 3.015 0.448 DKFZJournal of Virology déc. 2001 p11913-11919 pHPV 87 E1 L1 11/2 A3 3.0152.794 DKFZ Journal of Virology déc. 2001 p11913-11919 pHPV 87 L1 E1 37A3 3.015 1.227 DKFZ Journal of Virology déc. 2001 p11913-11919 pHPV 87E11 E2 as A3 3.015 1.130 DKFZ Journal of Virology déc. 2001 p11913-11919pHPV 58 A8 3.800 7.824 DKFZ Virology (1990) 177: 833-836 pHPV 59 A72.695 7.896 DKFZ Int. J. Cancer (1995) 61: 13-22 pHPV 87 A9 2.695 7.801DKFZ Int. J. Cancer (1995) 61: 13-22 pHPV 81 A3 3.204 4.759 DKFZVirology (2001) 283: 139-147 pHPV 82 A5 3.204 7.871 DKFZ Clin. Diagn.Lab. Immu. (2000) 7: 91-95 pHPV 85 A7 3.500 7.812 DKFZ Journal ofGeneral Virology (1999) 80, 2923-2929 DKFZ is DeuschesKresbsorschungszentrum; Tumorvirologie; ATV0660; Im Neuenheimer Feld242; DE-Heidelberg; Germany; CNCM is Collection Nationale de Cultures deMicroorganisme; Institut Pasteur; 25, rue du Docteur Roux; F-75724 ParisCedex 15; France; ATCC is American Type Culture Collection; 10801University Blvd.; Manassas, Virginia 20110-2209; U.S.A. All HPV strainsare available from DKFZ.

TABLE 52 A5 and A6 Systems PCR simplex probe conditions Kit KitQuantitect probe PCR MgCl₂ 6 mM Plasmid concentration 10⁸ cop deplasmides/PCR forward reverse primer primer probes Thermoprofile Name μMName μM Name μM A5 System A. B. C. D. E: PCR simplex probe conditionsSystem A 55° C. A5E6f1 0.4 A5E6r1 0.4 A5E6S1 0.3 System A 53° C. A5E6f10.4 A5E6r1 0.4 A5E6S1b 0.2 System B 55° C. A5E6f2 0.3 A5E6r2 0.3 A5E6S20.4 System C 55° C. A5E6f3 0.6 A5E6r3 0.6 A5E6S3 0.3 System D 56° C.A5E6f4 0.3 A5E6r4 0.3 A5E6S4 0.3 System E 55° C. A5E6f5 0.6 A5E6r5 0.6A5E6S4 0.2 A6 System A. B. C. D. E: PCR simplex probe conditions SystemA 58° C. A6E6f1 0.4 A6E6r1 0.4 A6E6S1 0.3 System B 57° C. A6E6f2 0.6A6E6r1 0.6 A6E6S1 0.4 System C 55° C. A6E6f3 0.6 A6E6r2 0.6 A6E6S2 0.3System C 57° C. A6E6f3 0.6 A6E6r2 0.6 A6E6S2b 0.2 System D 58° C. A6E6f40.5 A6E6r1 0.5 A6E6S1 0.4 System E 57° C. A6E6f5 0.4 A6E6r3 0.4 A6E6S30.3 System E 56° C. A6E6f5 0.4 A6E6r3 0.4 A6E6S3b 0.2

TABLE 53 A5 System A, B, C, D, E, specificity

*Plasmid concentration: 5 · 10⁵ copies of plasmids/PCR ND: no DetectionNT: no test

TABLE 54 A6 System A, B, C, D, E, specificity

*Plasmid concentration: 5 10^(b) copies of plasmids/PCR ND: no DetectionNT: no test

TABLE 55 A7 System A. B. C. D: PCR simplex probe and PCR multiplexprobes conditions Kit Kit Quantitect probe PCR MgCl₂ 6 mM Plasmidconcentration 10⁶ cop de plasmides/PCR forward primer reverse primerprobes Thermoprofile Name μM Name μM Name μM System A 53° C. A7E16f1a0.3 A7E16r1b 0.2 A7E1ZAS61f 0.2 A7E16f2a 0.3 A7E16r2b 0.3 A7E1S63f 0.2A7E16f3a 0.3 A7E16r3b 0.3 A7E1S64f 0.2 A7E1S40f 0.2 A7E1ZBS74f 0.2System B 52° C. A7E115f1a 0.3 A7E115r1a 0.2 A7E1ZBS26f 0.2 A7E115f2a 0.3A7E115r2b 0.3 A7E1ZBS74f 0.2 A7E115f3d 0.3 A7E1ZBS80f 0.2 System C 51°C. A7E17f1 0.4 A7E17r1 0.3 A7E1ZCS11f 0.2 A7E17f2 0.4 A7E17r2 0.4A7E1ZCS45f 0.2 A7E17f3 0.4 A7E1ZCS63f 0.2 A7E1ZCS90f 0.2 System D 53° C.A7E12f1 0.3 A7E12r2 0.3 A7E1S36f 0.2 A7E12f2 0.3 A7E12r3 0.1 A7E1S37f0.2 A7E1S38f 0.2 A7E1ZDS2f 0.2

TABLE 56 A7 System A. specificity

ND: no Detection NT: no test

TABLE 57 A7 System B. specificity

ND: no Detection NT: no test

TABLE 58 A7 System C. specificity

ND: no Detection NT: no test

TABLE 59 A7 System D. specificity

ND: no Detection NT: no test

TABLE 60 A9 Systems: PCR simplex and PCR multiplex probes conditions KitKit Quantitect probe PCR MgCl₂ 5 mM Plasmid concentration 10⁶ cop/PCRforward primer reverse primer probes Thermoprofile Name μM Name μM NameμM System C 51° C. A9E1f8 0.4 A9E1r5 0.4 A9E1S10a 0.2 A9E1f10 0.4 A9E1r60.4 A9E1S12a 0.2 A9E1f12 0.2 System E2 52° C. A9E2f1a 0.4 A9E2r1 0.4A9E2Z7S1 0.2 A9E2f2a 0.6 A9E2r2 0.4 A9E2Z7S2 0.2 A9E2f4a 0.4 A9E2r4 0.6A9E2Z7S3a 0.2 A9E2r13 0.4 A9E2Z7S4a 0.2 A9E2r14 0.4 System E3 52° C.A9E2f1a 0.4 A9E21r1cz 0.4 A9E2Z7S1 0.2 A9E2f2a 0.4 A9E21r2az 0.4A9E2Z7S2 0.2 A9E2f4a 0.4 A9E21r3az 0.4 A9E2Z7S3a 0.2 A9E21r4fz A9E2Z7S4a0.2 A9E21r5az System E4 52° C. A9E2Z5Z6f1c 0.4 A9E21r1cz 0.4 A9E2Z7S10.2 A9E2Z5Z6f2c 0.4 A9E21r2az 0.4 A9E2Z7S2 0.2 A9E2Z5Z6f3b 0.4 A9E21r3az0.4 A9E2Z7S3a 0.2 A9E21r4fz A9E2Z7S4a 0.2 A9E21r5az System F 52° C.A9E2-1f1az 0.4 A9E2-1r1cz 0.4 A9E2Z7S1 0.2 A9E2-1f2bz 0.5 A9E2-1r2az 0.4A9E2Z7S2 0.2 A9E2-1f3dz 0.4 A9E2-1r3az 0.4 A9E2Z7S3a 0.2 A9E2-1f4cz 0.4A9E2-1r4fz 0.4 A9E2Z7S4a 0.2 A9E2-1r5az 0.4 System G Z7 52° C.A9E2-1f1az 0.4 A9E2r8 0.4 A9E2Z7S1 0.2 A9E2-1f2bz 0.4 A9E2r10 0.4A9E2Z7S2a 0.2 A9E2-1f3dz 0.4 A9E2r12 0.4 A9E2Z7S3a 0.2 A9E2-1f4cz 0.5A9E2r12B 0.4 A9E2Z7S4a 0.2 A9E2r15 0.4 System G Z8 52° C. A9E2-1f1az 0.6A9E2r8 0.4 A9E2Z8S2f 0.2 A9E2-1f2bz 0.4 A9E2r10 0.4 A9E2Z8S61f 0.2A9E2-1f3dz 0.4 A9E2r12 0.4 A9E2Z8S127f 0.2 A9E2-1f4cz 0.5 A9E2r12B 0.4A9E2Z8S156f 0.2 A9E2r15 0.6 A9E2Z8S210f 0.2 A9E2Z8S250f 0.2 System H 53°C. A9E2f6 0.4 A9E2r10 0.4 A9E2Z8S2f 0.2 A9E2f8 0.4 A9E2r12B 0.4A9E2Z8S61f 0.2 A9E2f9 0.4 A9E2r15 0.4 A9E2Z8S127f 0.2 A9E2r16 0.4A9E2Z8S156f 0.2 A9E2Z8S210f 0.2 A9E2Z8S231f 0.2 A9E2Z8S250f 0.2

TABLE 61 A9 System C specificity

*Probes tested only with some A9 plasmids (i.e.. those A9 plasmids.which are indicated in this table) ND: not detected NT: not tested

TABLE 62 A9 System E2 specificity

ND: not detected NT: not tested

TABLE 63 A9 System E3 specificity

ND: not detected NT: not tested

TABLE 64 A9 System E4 specificity

ND: not detected NT: not tested

TABLE 65 A9 System F specificity

ND: not detected NT: not tested

TABLE 66 A9 System G Z7 specificity

ND: not detected NT: not tested

TABLE 67 A9 system G Z8 specificity

ND: not detected NT: not tested *probe at 0.1 μM in these tests **probesat 0.3 μM in this test

TABLE 68 A9 System H specificity

ND: not detected NT: not tested

TABLE 69 A5 Systems A, B, C, D, E/sensitivity System A System B System CA5E6S1/HPV51 A5E6S1b/HPV51 A5E6S2/HPV51 A5E6S3/HPV51 Moy Ct Moy RFUs MoyCt Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10³ cop/PCR 27.95 2700 25.651100 27.3 180 24.3 430 10² cop/PCR 31.3 2500 29.75 1000 31.0 180 26.85380  10 cop/PCR 34.85 2200 34.3 700 35.7 160 30.0 280   1 cop/PCR 36.951700 36.65 450 38.85 150 33.1 180 H2O sample ND ND ND ND ND ND ND ND ADNSample ND ND ND ND ND ND ND ND r2/slope/PCR efficiency0.997/−2.988/116.1% 0.997/−3.647/88% 0.990/−3.924/79.8%0.997/−2.955/118% System D System E Copy number HPV A5E6S4/HPV51A5E6S4/HPV51 plasmid/PCR Moy Ct Moy RFUs Moy Ct Moy RFUs 10³ cop/PCR31.45 750 24.55 500 10² cop/PCR 35.1 700 27.45 450 ^( 10 cop/PCR) 39.7500 31.15 450   1 cop/PCR 43.2 250 33.75 500 H2O sample ND ND ND ND ADNSample ND ND ND ND r2/slope/PCR efficiency 0.994/−3.990/78.1%0.997/−3.128/108.8% ND: No Detection NT: Not tested

TABLE 70 A6 Systems A, B, C, D, E/sensitivity System A System B System CCopy number HPV A6E6S1/HPV56 A6E6S1/HPV56 A6E6S2/HPV56 A6E6S2b/HPV56plasmid/PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct MoyRFUs 10³ cop/PCR 27.25 400 27.1 600 33.955 1700 29.55 650 10² cop/PCR27.8 450 27.9 700 35.78 1500 33.1 550  10 cop/PCR 30.8 450 30.75 70038.89 1200 38.25 300   1 cop/PCR 34.25 350 34.15 450 43.09 600 ND ND H2Osample ND ND ND ND ND ND ND ND ADN Sample ND ND ND ND ND ND ND NDr2/slope/PCR 0.974/−3.228/104.1% 0.997/−3.16/107% 0.989/−3.270/102.2%0.998/−3.699/86.4% efficiency System D System E Copy number HPVA6E6S1/HPV56 A6E6S3/HPV56 A6E6S3b/HPV56 plasmid/PCR Moy Ct Moy RFUs MoyCt Moy RFUs Moy Ct Moy RFUs 10³ cop/PCR 28.95 750 26.1 600 27.5 750 10²cop/PCR 30.75 750 27.45 650 30.35 750  10 cop/PCR 33.65 800 30.45 55033.9 600   1 cop/PCR 35.4 700 32.15 550 ND ND H2O sample ND ND ND ND NDND ADN Sample ND ND ND ND ND ND r2/slope/PCR 0.987/−2.461/154.8%0.986/−2.126/195.4% 0.990/−3.183/106.1% efficiency ND: No detection NT:Not tested

TABLE 71 A7 System A, sensitivity copy number HPV plasmid/ A7E1ZAS61fHPV 18 A7E1S63f HPV 59 A7E1S64f HPV 39 A7E1S40f HPV 45 PCR Moy Ct MoyRFU Moy Ct Moy RFUs Moy Ct Moy RFUs Ecart-type Moy RFUs 10⁶ cop/PCR 22.7165 21.7 125 22.1 240 24.6 305 10⁵ cop/PCR 25.95 170 24.6 95 25.0 31027.45 320 10⁴ cop/PCR 29.15 180 27.1 125 28.3 320 30.6 310 10³ cop/PCR32.6 180 31.0 105 31.1 350 33.85 272.5 10² cop/PCR 35.2 170 33.95 7034.15 290 36.9 282.5 H₂O sample ND ND ND ND ND ND ND ND ADN sample ND NDND ND ND ND ND ND r2/slope/efficiency PCR 0.997/−3.16/107%0.993/−3.1/110% 0.996/−3.19/106% 0.995/−3.27/102% copy number HPVplasmid/ A7E1ZBS74f HPV 68 A7E1ZBS74f HPV 39 PCR Moy Ct Moy RFUs Moy CtMoy RFUs 10⁶ cop/PCR 24.2 597.585 22.5 1095.195 10⁵ cop/PCR 26.85 611.5225.25 1182.205 10⁴ cop/PCR 31.15 557.525 28.05 1062.505 10³ cop/PCR35.35 496.425 32.45 1005.26 10² cop/PCR 39.5 473.395 40.6 804 H₂O sampleND ND ND ND ADN sample ND ND ND ND r2/slope/efficiency PCR0.995/−3.919/80% 0.972/−3.271/102.2% ND: no Detection NT: no test

TABLE 72 A7 System B. sensitivity copy number HPV plasmid/ A7E1ZBS26fHPV 18 A7E1ZBS26f HPV 45 A7E1ZBS74f HPV 68 A7E1ZBS74f HPV 39 PCR Moy CtMoy RFU Moy Ct Moy RFUs Moy Ct Moy RFUs Ecart-type Moy RFUs 10⁶ cop/PCR21.35 222.5 22.1 115 21.2 705 24.85 892.5 10⁵ cop/PCR 23.05 252.5 24.75130 24.7 690 27.0 852.5 10⁴ cop/PCR 26.5 225 28.05 115 28.15 645 31.2632.5 10³ cop/PCR 28.6 180 29.7 112.5 30.85 540 32.65 445 10² cop/PCR31.35 155 30.85 112.5 33.25 390 31.85 660.5 H₂O sample ND ND ND ND 42.564 42.5 64 ADN sample ND ND ND ND ND 23 ND 23 r2/slope/efficiency PCR0.987/−2.55/146% 0.970/−2.24/179% 0.995/−3.02/114% 0.887/−1.96/223% copynumber HPV plasmid/ A7E1ZBS80f HPV 59 A7E1ZBS79f HPV 59 A7E1ZBS27f HPV45 A7E1ZBS27f HPV 18 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs 10⁶ cop/PCR 19.65 952.5 18.85 387.5 20.3 675 20.9 112.510⁵ cop/PCR 21.85 1025 21.5 427.5 23.25 595 24.35 117.5 10⁴ cop/PCR23.75 997.5 24.1 405 26.3 557.5 28 122.5 10³ cop/PCR 27.1 952.5 26.25412.5 27.8 502.5 31.7 137.5 10² cop/PCR 31.7 907.5 29.0 377.5 33.05 44031.05 157.5 H₂O sample ND ND ND ND ND ND ND ND ADN sample ND ND ND ND NDND ND ND r2/slope/efficiency PCR 0.981/−2.94/119% 0.976/−2.51/150%0.973/−3.01/115% 0.934/−2.718/133.3% ND: no Detection NT: no test

TABLE 73 A7 System C. sensitivity copy number HPV plasmid/A7E1ZCS11f/HPV 45 A7E1ZCS45f/HPV 39 A7E1ZCS63f/HPV 59 A7E1ZCS90f/HPV 68PCR Moy Ct Moy RFU Moy Ct Moy RFUs Moy Ct Moy RFUs Ecart-type Moy RFUs10⁶ cop/PCR 21.95 349.45 20.35 649.815 23.1 256.71 24.05 234.305 10⁵cop/PCR 24.7 344.81 23.4 623.445 26.4 224.625 27.0 200 10⁴ cop/PCR 28.35274.81 26.95 502.52 28.9 178.505 30.5 140.205 10³ cop/PCR 31.6 154.56530.35 364.095 33.0 107.145 33.4 92.505 10² cop/PCR 35.1 48.72 33.45173.17 35.3 49.965 37.15 46.65 H₂O sample ND ND ND ND ND ND ND ND ADNsample ND ND ND ND ND ND ND ND r2/slope/efficiency PCR 0.997/−3.321/100%0.998/−3.322/100% 0.993/−3.111/109.9% 0.998/−3.255/102.9% copy numberHPV plasmid/ A7E1ZCS11f/HPV 18 A7E1ZCS40f/HPV 18 A7E1ZCS40f/HPV 45A7E1ZCS40f/HPV 59 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs 10⁶ cop/PCR 22.05 247.645 23.6 65.31 23.65 161.135 22.259.985 10⁵ cop/PCR 24.45 230.285 26.25 79.255 27.5 138.725 22.65 27.1910⁴ cop/PCR 27.1 197.085 29.95 56.015 29.85 107.025 28.7 11.595 10³cop/PCR 31.35 94.76 34.2 29.31 37.9 14 N/A ND 10² cop/PCR 35.0 12.61 N/AND N/A ND N/A ND H₂O sample ND ND ND ND ND ND ND ND ADN sample ND ND NDND ND ND ND ND r2/slope/efficiency PCR 0.993/−3.275/102%0.986/−3.55/91.3% 0.988/−3.079/111.3% 0.922/−4.269/71.5% ND: noDetection NT: no test

TABLE 74 A7 System D. sensitivity copy number HPV plasmid/ A7E1ZDS36fHPV 45 A7E1ZDS37f HPV 18 A7E1ZDS38f HPV 59 A7E1ZDS38f HPV 39 PCR Moy CtMoy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR26.0 705 21.9 305 21.05 525 24.2 190 10⁵ cop/PCR 29.5 645 25.55 302.525.35 487.5 27.8 192.5 10⁴ cop/PCR 33.25 537.5 27.1 292.5 29.2 402.532.25 155 10³ cop/PCR 36.95 440 30.95 265 32.5 335 35.95 147.5 10²cop/PCR 40.0 332.5 32.45 247.5 33.7 342.5 39.2 140 H₂O sample ND ND NDND ND ND ND ND ADN sample ND ND ND ND ND ND ND ND r2/slope/efficiencyPCR 0.998/−3.622/88.8% 0.978/−2.652/138.3% 0.981/−3.253/103%0.995/−3.805/83.2% copy number HPV plasmid/ A7E1ZDS2f HPV 68 A7E1ZDS2fHPV 39 A7E1ZDS3f HPV 45 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct MoyRFUs 10⁶ cop/PCR 23.1 942 21.15 113.5 21.85 2075 10⁵ cop/PCR 26.8 898.525.55 97.5 24.9 1875 10⁴ cop/PCR 30.5 842.5 28.8 87.5 27.75 1617.5 10³cop/PCR 33.15 677.5 32.25 79 31.05 1250 10² cop/PCR 36.65 635 31.75 8034.2 1007.5 H₂O sample ND ND ND ND ND ND ADN sample ND ND 36.1 ND ND NDr2/slope/efficiency PCR 0.995/−3.334/99.5% 0.941/−2.797/127.8%0.999/−3.086/110.9% ND: no Detection NT: no test

TABLE 75 A9 System C, sensitivity copy number HPV plasmid/ A9E1S10a/HPV16 A9E1S10a/HPV 31 A9E1S10a/HPV 33 A9E1S10a/HPV 35 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 22.4 457.522.15 477 22.5 410 24.4 233.5 10⁵ cop/PCR 27.2 402.5 26.45 360 28.2 34429.0 202 10⁴ cop/PCR 30.85 341 29.9 342 31.0 306.5 32.65 192 10³ cop/PCR34.3 322.5 33.2 316.5 34.35 272 36.25 167 10² cop/PCR 37.35 320 36.7259.5 37.1 227.5 39.45 135.5 H₂O sample ND ND ND ND ND ND ND ND ADNsample ND ND ND ND ND ND ND ND r2/slope/efficiency PCR0.996/−3.705/86.2% 0.998/−3.586/90.0% 0.987/−3.536/91.8%0.997/−3.735/85.2% copy number HPV plasmid/ A9E1S10a/HPV 52 A9E1S10a/HPV58 A9E1S10a/HPV 67 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs10⁶ cop/PCR 21.85 650 23.65 620 22.0 351 10⁵ cop/PCR 26.4 507 28.3 558.525.95 339 10⁴ cop/PCR 29.9 474.5 32.3 484 29.75 304 10³ cop/PCR 33.55429.5 35.95 422.5 33.05 261.5 10² cop/PCR 36.7 350.5 39.35 271.5 36.45222.5 H₂O sample ND ND ND ND ND ND ADN sample ND ND ND ND ND NDr2/slope/efficiency PCR 0.997/−3.680/87.0% 0.997/−3.915/80.1%0.999/−3.594/89.8% copy number HPV plasmid/ A9E1S12a/HPV 31 A9E1S12a/HPV35 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 26.45 61 22.45 50410⁵ cop/PCR 31.3 53.5 27.6 469.5 10⁴ cop/PCR 32.2 67 31.3 452 10³cop/PCR 36.25 56 34.25 419.5 10² cop/PCR 39.45 51 37.7 356.5 H₂O sampleND ND ND ND ADN sample ND ND ND ND r2/slope/efficiency PCR0.971/−3.099/110.2% 0.994/−3.719/85.7% ND: not detected NT: not tested

TABLE 76 A9 System E2, sensitivity copy number HPV plasmid/ A9E2Z7S1/HPV16 A9E2Z7S1/HPV 31 A9E2Z7S1/HPV 33 A9E2Z7S1/HPV 52 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 19.8 79523.05 485 31.35 88.5 24.25 86 10⁵ cop/PCR 24.1 765 26.5 425 34.4 83.527.05 82.5 10⁴ cop/PCR 27.3 705 29.8 385 38.1 65 30.8 78.5 10³ cop/PCR31.5 610 33.0 342.5 42.8 35 33.85 77.5 10² cop/PCR 34.95 287.5 37.25177.5 38.9 41.5 36.2 82.5 H₂O sample ND ND ND ND ND ND ND ND ADN sampleND ND ND ND ND ND ND ND r2/slope/efficiency PCR 0.996/−3.767/84.3%0.998/−3.492/93.4% 0.869/−2.762/130.2% 0.996/−3.069/111.7% copy numberHPV plasmid/ A9E2Z7S1/HPV 58 A9E2Z7S1/HPV 67 A9E1Z7S2/HPV 33A9E1Z7S2/HPV 58 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs MoyCt Moy RFUs 10⁶ cop/PCR 23.4 145 29.35 120.5 26.4 1257.5 20.95 485 10⁵cop/PCR 26.4 140 32.85 105 30.0 1120 24.35 395 10⁴ cop/PCR 30.45 12536.9 77.5 33.6 877.5 28.15 340 10³ cop/PCR 34.35 95 42.4 37.5 37.05 52031.85 310 10² cop/PCR 37.05 91 ND ND 40.0 107.5 34.7 257.5 H₂O sample NDND ND ND ND ND ND ND ADN sample ND ND ND ND ND ND ND NDr2/slope/efficiency PCR 0.99/−3.525/90.2% 0.991/−4.333/70.1%0.993/−3.431/95.7% 0.999/−3.508/92.8% copy number HPV plasmid/A9E1Z7S2/HPV 52 A9E1Z7S2/HPV 67 A9E1Z7S3a/HPV 35 PCR Moy Ct Moy RFUs MoyCt Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 21.1 182.5 26.6 473 27.25 82510⁵ cop/PCR 24.65 197.5 30.4 430 30.85 807.5 10⁴ cop/PCR 28.15 192.533.2 390 34.5 505 10³ cop/PCR 31.85 182.5 35.7 200 37.9 82.5 10² cop/PCR34.95 162.5 40.3 115 40.4 47.5 H₂O sample ND ND ND ND ND ND ADN sampleND ND ND ND ND ND r2/slope/efficiency PCR 0.999/−3.480/92.8%0.989/−3.279/101.8% 0.990/−3.396/97% copy number HPV plasmid/A9E2Z7S4a/HPV 31 A9E2Z7S4a/HPV 52 A9E2Z7S4a/HPV 16 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 23.2 460 21.45 485 21.85182.5 10⁵ cop/PCR 26.5 452.5 24.5 425 25.6 177.5 10⁴ cop/PCR 29.85 43528.35 385 29.8 147.5 10³ cop/PCR 34.05 322.5 31.65 340 33.75 135 10²cop/PCR 38.0 165 35.55 280 36.2 145 H₂O sample ND ND ND ND ND ND ADNsample ND ND ND ND ND ND r2/slope/efficiency PCR 0.998/−3.720/85.7%0.996/−3.517/92.5% 0.996/−3.687/86.7% copy number HPV plasmid/A9E2Z7S4a/HPV 33 A9E2Z7S4a/HPV 58 A9E2Z7S4a/HPV 67 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 32.0 57.5 23.6 115 28.45177.5 10⁵ cop/PCR 35.0 57.5 27.2 122.5 32.0 140 10⁴ cop/PCR 43.6 31 31.587.5 36.3 107.5 10³ cop/PCR ND ND 34.1 90 42.8 42.5 10² cop/PCR ND ND40.4 50 46.7 20 H₂O sample ND ND ND ND ND ND ADN sample ND ND ND ND NDND r2/slope/efficiency PCR 0.938/−5.797/48..8% 0.980/−4.047/76.6%0.981/−4.736/62.6% ND: not detected NT: not tested

TABLE 77 A9 System E3, sensitivity copy number HPV plasmid/ A9E2Z7S1/HPV16 A9E2Z7S1/HPV 31 A9E2Z7S1/HPV 33 A9E2Z7S1/HPV 52 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 18.0 72519.7 457 22.8 69 20.3 59 10⁵ cop/PCR 20.3 754 23.7 428 26.9 58 22.7 7710⁴ cop/PCR 23.3 692 25.5 390 30.2 55 24.7 97 10³ cop/PCR 26.2 611 29.4322 34.1 48 28.4 73 10² cop/PCR 30.0 476 32.6 208 37.2 29 32.2 65 H²Osample ND ND ND ND ND ND ND ND ADN sample ND ND ND ND ND ND ND NDr2/slope/efficiency PCR 0.994/−2.994/115.8% 0.994/−3.148/107.8%0.988/−3.647/88% 0.987/−2.952/118.2% copy number HPV plasmid/A9E2Z7S1/HPV 58 A9E2Z7S1/HPV 67 A9E1Z7S2/HPV 33 A9E1Z7S2/HPV 58 PCR MoyCt Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR20.8 117 27.5 111 16.7 1741 20.8 636 10⁵ cop/PCR 22.5 113 29.4 110 19.21767 22.5 537 10⁴ cop/PCR 24.9 123 33.1 99 22.7 1574 24.9 693 10³cop/PCR 28.4 117 36.9 70 26.9 1473 28.4 465 10² cop/PCR 31.3 104 41.3 3029.6 1172 31.3 357 H₂O sample ND ND ND ND ND ND ND ND ADN sample ND NDND ND ND ND ND ND r2/slope/efficiency PCR 0.987/−2.296/134.9%0.990/−3.509/92.7% 0.995/−3.318/100.1% 0.772/−2.474/153.6% copy numberHPV plasmid/ A9E1Z7S2/HPV 52 A9E1Z7S2/HPV67 A9E1Z7S3a/HPV 35 PCR Moy CtMoy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 18.6 393 25.3 43325.3 869 10⁵ cop/PCR 21.1 303 28.8 375 27.6 611 10⁴ cop/PCR 25.1 27431.8 299 31.6 509 10³ cop/PCR 28.0 260 35.4 214 34.7 294 10² cop/PCR32.4 152 ND ND ND ND H₂O sample ND ND ND ND ND ND ADN sample ND ND ND NDND ND r2/slope/efficiency PCR 0.994/−3.523/92.3% 0.997/−3.323/99.9%0.993/−3.328/104.1% copy number HPV plasmid/ A9E2Z7S4a/HPV 31A9E2Z7S4a/HPV 52 A9E2Z7S4a/HPV 16 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs 10⁶ cop/PCR 19.2 494 18.1 427 18.9 79 10⁵ cop/PCR 22.8451 20.2 473 21.0 199 10⁴ cop/PCR 26.0 360 23.7 460 23.7 170 10³ cop/PCR29.5 283 27.5 419 27.2 166 10² cop/PCR 33.4 82 31.0 273 31.3 37 H₂Osample ND ND ND ND ND ND ADN sample ND ND ND ND ND NDr2/slope/efficiency PCR 0.998/−3.510/92.7% 0.995/−3.312/100.4%0.989/−3.006/115.1% copy number HPV plasmid/ A9E2Z7S4a/HPV 33A9E2Z7S4a/HPV 58 A9E2Z7S4a/HPV 67 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs 10⁶ cop/PCR 21.0 66 19.7 105 27.2 494 10⁵ cop/PCR 24.262 22.8 108 30.5 451 10⁴ cop/PCR 27.5 56 25.8 108 36.1 360 10³ cop/PCR30.8 53 30.3 78 45.4 283 10² cop/PCR ND 22 35.6 57 ND ND H₂O sample NDND ND ND ND ND ADN sample ND ND ND ND ND ND r2/slope/efficiency PCR0.994/−3.257/102.8% 0.976/−3.920/79.9% 0.959/−5.655/50.3% ND: notdetected NT: not tested

TABLE 78 A9 System E4, sensitivity copy number HPV plasmid/ A9E2Z7S1/HPV16 A9E2Z7S1/HPV 31 A9E2Z7S1/HPV 52 A9E2Z7S1/HPV 33 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 19.8 91521.45 795 24.25 65 24.35 105 10⁵ cop/PCR 23.25 940 24.3 717.5 31.1 47.528.65 90 10⁴ cop/PCR 26.75 840 28.25 540 37.8 27 37.8 83 10³ cop/PCR30.85 615 32.5 412.5 35.15 40 36.15 75 10² cop/PCR 34.95 507.5 35.0272.5 37.3 37.5 38.95 47.5 H₂O sample ND ND ND ND ND ND ND ND ADN sampleND ND ND ND ND ND ND ND r2/slope/efficiency PCR 0.993/−3.789/83.7%0.996/−3.509/92.8% 0.889/−3.017/114.5% 0.996/−3.664/87.5% copy numberHPV plasmid/ A9E2Z7S1/HPV 58 A9E2Z7S1/HPV 67 A9E1Z7S2/HPV 33A9E1Z7S2/HPV 58 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs MoyCt Moy RFUs 10⁶ cop/PCR 23.8 157.5 22.9 207.5 22.0 2871.625 22.75 903.9210⁵ cop/PCR 27.6 167.5 26.45 190 25.7 2916.84 25.95 822.335 10⁴ cop/PCR37.8 128 37.8 160 29.95 2525.075 29.55 731.815 10³ cop/PCR 36.0 90 34.25122.5 33.55 1574.41 33.95 614.88 10² cop/PCR 39.8 45 39.8 52.5 37.451675.46 37.35 155.27 H²O sample ND ND ND ND ND ND ND ND ADN sample ND NDND ND ND ND ND ND r2/slope/efficiency PCR 0.999/−4.075/75.9%0.996/−4.075/75.9% 0.998/−3.866/81.4% 0.998/−3.725/85.5% copy number HPVplasmid/ A9E1Z7S2/HPV 52 A9E1Z7S2/HPV67 A9E1Z7S3a/HPV 35 PCR Moy Ct MoyRFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 20.4 372.5 21.3 122522.6 1085.81 10⁵ cop/PCR 23.6 360 24.35 1157.5 26.1 1037.5 10⁴ cop/PCR27.2 345 27.9 1055 29.6 932.32 10³ cop/PCR 30.85 347.5 31.75 935 33.75730.08 10² cop/PCR 34.65 310 35.85 757.5 38.55 501.76 H₂O sample ND NDND ND ND ND ADN sample ND ND ND ND ND ND r2/slope/efficiency PCR0.997/−3.586/90.1% 0.998/−3.641/88.2% 0.997/−3.960/78.9% copy number HPVplasmid/ A9E2Z7S4a/HPV 31 A9E2Z7S4a/HPV 52 A9E2Z7S4a/HPV 16 PCR Moy CtMoy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 21.8 800 20.45 64022.35 267.5 10⁵ cop/PCR 25.2 747.5 24.0 600 25.0 260 10⁴ cop/PCR 28.4665 27.5 562.5 29.55 240 10³ cop/PCR 33.3 505 30.95 532.5 32.75 210 10²cop/PCR 37.2 335 34.4 422.5 33.55 207.5 H₂O sample ND ND ND ND ND ND ADNsample ND ND ND ND ND ND r2/slope/efficiency PCR 0.996/−3.872/81.2%1/−3.476/94% 0.976/−3.010/114.9% copy number HPV plasmid/ A9E2Z7S4a/HPV33 A9E2Z7S4a/HPV 58 A9E2Z7S4a/HPV 67 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs 10⁶ cop/PCR 24.45 212.5 24.2 340 23.25 415 10⁵ cop/PCR27.3 230 27.45 305 25.55 407.5 10⁴ cop/PCR 31.1 205 31.2 250 30.5 312.510³ cop/PCR 35.1 162.5 34.35 187.5 33.75 277.5 10² cop/PCR 36.8 127.538.9 117.5 38.2 145 H₂O sample ND ND ND ND ND ND ADN sample ND ND ND NDND ND r2/slope/efficiency PCR 0.991/−3.264/102.5% 0.998/−3.632/88.5%0.989/−3.810/83% ND: not detected NT: not tested

TABLE 79 A9 System F, sensitivity copy number HPV plasmid/ A9E2Z7S1/HPV16 A9E2Z7S1/HPV 31 A9E2Z7S1/HPV 33 A9E2Z7S1/HPV 52 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 19.45 181519.85 1227.5 23.85 63 25.25 40 10⁵ cop/PCR 22.25 1727.5 22.95 1215 27.163 28.55 41.5 10⁴ cop/PCR 26.1 1665 26.75 1150 30.55 62.5 31.6 37.5 10³cop/PCR 26.65 1535 29.6 1055 33.9 62 35.4 41 10² cop/PCR 33.15 1312.535.2 753.5 37.8 47.5 38.25 34 H₂O sample ND ND ND ND ND ND ND ND ADNsample ND ND ND ND ND ND ND ND r2/slope/efficiency PCR0.969/−3.202/105.2% 0.990/−3.739/85.1% 0.999/−3.487/93.5%0.998/−3.651/87.9% copy number HPV plasmid/ A9E2Z7S1/HPV 58 A9E2Z7S1/HPV67 A9E1Z7S2/HPV 33 A9E1Z7S2/HPV 58 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 22.8 132.5 22.85 138 20.23013.235 21.75 1330.945 10⁵ cop/PCR 26.1 125 25.95 127.5 23.4 3085.824.85 1292.96 10⁴ cop/PCR 29.55 120 29.6 120 26.7 2967.135 28.351266.585 10³ cop/PCR 33.45 107 33.65 96 30.45 2499.875 32.25 973.095 10²cop/PCR 37.4 100 35.85 100 33.8 2129.57 35.55 841.57 H₂O sample ND ND NDND ND ND ND ND ADN sample ND ND ND ND ND ND ND ND r2/slope/efficiencyPCR 0.998/−3.651/87.9% 0.993/−3.366/98.20% 0.999/−3.435/95.5%0.999/−3.502/93% copy number HPV plasmid/ A9E1Z7S3a/HPV 35 A9E2Z7S4a/HPV31 A9E2Z7S4a/HPV 52 A9E2Z7S4a/HPV 58 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 22.1 1337.995 21.85 70521.75 1022.5 22.1 522.5 10⁵ cop/PCR 25.05 1354.145 24.1 730 24.25 992.524.55 460 10⁴ cop/PCR 29.4 1254.22 27.9 665 27.65 1002.5 28.9 455 10³cop/PCR 33.25 1018.5 30.5 560 31.05 957.5 31.5 465 10² cop/PCR 37.0678.815 35.25 430 34.7 650 35.2 382.5 H₂O sample ND ND ND ND ND ND ND NDADN sample ND ND ND ND ND ND ND ND r2/slope/efficiency PCR0.996/−3.799/83.3% 0.989/−3.332/99.6% 0.996/−3.273/102.1%0.994/−3.317/100.2% copy number HPV plasmid/ A9E2Z7S4a/HPV 67A9E2Z7S4a/HPV 16 A9E2Z7S4a/HPV 33 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs 10⁶ cop/PCR 22.15 570 21.95 195 22.45 240 10⁵ cop/PCR24.2 670 24.65 227.5 25.15 245 10⁴ cop/PCR 28.75 555 29.2 196 29.7 25510³ cop/PCR 31.2 460 32.6 187.5 32.55 187.5 10² cop/PCR 32.7 447.5 35.7185 36.45 165 H₂O sample ND ND ND ND ND ND ADN sample ND ND ND ND ND NDr2/slope/efficiency PCR 0.964/−2.805/127.3% 0.994/−3.487/93.5%0.995/−3.538/91.7% ND: not detected NT: not tested

TABLE 80 A9 System G Z7, sensitivity copy number HPV plasmid/A9E2Z7S1/HPV 16 A9E2Z7S1/HPV 31 A9E2Z7S1/HPV 33 A9E2Z7S1/HPV 52 PCR MoyCt Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR20.25 771 20.85 468 22.85 113 23.2 82 10⁵ cop/PCR 23.55 713.5 24.5 419.526.4 119 26.45 75 10⁴ cop/PCR 27.0 654 27.6 375.5 30.8 79.5 29.65 65 10³cop/PCR 30.75 527 31.15 289 33.6 71 33.35 48.5 10² cop/PCR 34.45 296.535.1 186 37.3 44.5 37.05 30.5 H₂O sample ND ND ND ND ND ND ND ND ADNsample ND ND ND ND ND ND ND ND r2/slope/efficiency PCR 0.999/−3.557/91%0.998/−3.520/92.3% 0.996/−3.612/89.2% 0.998/−3.452/94.9% copy number HPVplasmid/ A9E2Z7S1/HPV 58 A9E2Z7S1/HPV 67 A9E1Z7S2a/HPV 33 A9E1Z7S2a/HPV58 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs10⁶ cop/PCR 22.15 187.5 21.8 207 19.8 1165 20.65 717 10⁵ cop/PCR 25.75165.5 24.9 199 23.1 1080.5 23.75 682 10⁴ cop/PCR 28.8 147 27.35 182.526.3 1015.5 27.35 603.5 10³ cop/PCR 32.45 100 32.05 128.5 30.05 844 30.9426 10² cop/PCR 35.35 63 35.4 78.5 33.65 605.5 34.8 234 H₂O sample ND NDND ND ND ND ND ND ADN sample ND ND ND ND ND ND ND ND r2/slope/efficiencyPCR 0.998/−3.313/100.4% 0.993/−3.425/95.9% 0.998/−3.469/94.2%0.998/−3.558/91% copy number HPV plasmid/ A9E1Z7S3a/HPV 35 A9E2Z7S4a/HPV31 A9E2Z7S4a/HPV 52 A9E2Z7S4a/HPV 58 PCR Moy Ct Moy RFUs Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 22.15 883.5 21.7 187.5 21.65257 22.35 145 10⁵ cop/PCR 25.3 795 25.95 180.5 26.45 268 26.05 136.5 10⁴cop/PCR 29.0 674.5 29.7 157.5 29.8 220 29.3 116 10³ cop/PCR 32.2 54633.4 128.5 33.6 155 33.65 80 10² cop/PCR 35.2 300 36.65 71 37.9 69 37.340 H₂O sample ND ND ND ND ND ND ND ND ADN sample ND ND ND ND ND ND ND NDr2/slope/efficiency PCR 0.999/−3.656/87.7% 0.999/−3.727/85.5%0.997/−3.967/78.7% 0.998/−3.751/84.7% copy number HPV plasmid/A9E2Z7S4a/HPV 67 A9E2Z7S4a/HPV 16 A9E2Z7S4a/HPV 33 PCR Moy Ct Moy RFUsMoy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 22.4 159 22.4 67 22.65 69.510⁵ cop/PCR 26.5 155.5 27.7 63 27.0 60 10⁴ cop/PCR 30.1 134 31.0 6030.25 58.5 10³ cop/PCR 34.35 90.5 34.6 46 33.4 51.5 10² cop/PCR 38.55 3338.4 23.5 37.8 32.5 H₂O sample ND ND ND ND ND ND ADN sample ND ND ND NDND ND r2/slope/efficiency PCR 0.996/−4.035/76.9% 0.996/−3.894/80.6%0.996/−3.670/87.3% ND: not detected NT: not tested

TABLE 81 A9 System G Z8, sensitivity copy number HPV plasmid/A9E2Z8S2f/HPV16 A9E2Z8S61f/HPV 31 A9E2Z8S127f/HPV 33 A9E2Z8S156f/HPV35PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶cop/PCR 20.9 275 20.8 439.5 20.4 357 21.85 347.5 10⁵ cop/PCR 24.65 274.525.25 382 24.95 322 25.75 334.5 10⁴ cop/PCR 27.85 270 29.1 328 28.75300.5 29.25 331.5 10³ cop/PCR 31.55 217 32.3 242 31.6 261.5 32.8 243 10²cop/PCR 35.3 107.5 36.3 86 37.0 98 36.45 121 H₂O sample ND ND ND ND NDND ND ND ADN sample ND ND ND ND ND ND ND ND r2/slope/efficiency PCR0.995/−3.587/90% 0.995/−3.799/83.3% 0.995/−3.990/78.1%0.997/−3.639/88.3% copy number HPV plasmid/ A9E2Z8S210f/HPV52A9E2AZ8S250f/HPV58 A9E2AZ8S250f/HPV67 PCR Moy Ct Moy RFUs Moy Ct MoyRFUs Moy Ct Moy RFUs 10⁶ cop/PCR 22.05 32 19.3 728 21.2 173 10⁵ cop/PCR25.65 57 22.6 663 26.2 152 10⁴ cop/PCR 27.45 54.5 26.0 728 29.6 157 10³cop/PCR 32.7 31 29.7 627 32.6 149 10² cop/PCR 37.75 14 32.9 457 36.6 98H₂O sample ND ND ND ND ND ND ADN sample ND ND ND ND ND NDr2/slope/efficiency PCR 0.976/−3.880/81% 0.999/−3.425/95.9%0.994/−3.701/86.3% ND: not detected NT: not tested

TABLE 82 A9 System H, sensitivity copy number HPV plasmid/A9E2Z8S2f/HPV16 A9E2Z8S61f/HPV 31 A9E2Z8S127f/HPV 33 A9E2Z8S156f/HPV35PCR Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶cop/PCR 20.55 567.5 20.55 570 20.25 550 21.2 435 10⁵ cop/PCR 23.7 53023.25 530 23.7 490 24.55 437.5 10⁴ cop/PCR 27.2 465 27.05 507.5 27.4482.5 28.1 375 10³ cop/PCR 31.85 395 29.8 400 31.5 450 32.95 250 10²cop/PCR 34.3 220 34.05 247.5 35.35 212.5 37.05 172.5 H₂O sample ND ND NDND ND ND ND ND ADN sample ND ND ND ND ND ND ND ND r2/slope/efficiencyPCR 0.99/−3.570/90.6% 0.995/−3.352/98.8% 0.988/−3.805/83.2%0.995/−4.011/77.5% copy number HPV plasmid/ A9E2Z8S210f/HPV52A9E2AZ8S250f/HPV58 A9E2AZ8S250f/HPV67 PCR Moy Ct Moy RFUs Moy Ct MoyRFUs Moy Ct Moy RFUs 10⁶ cop/PCR 21.65 240 20.15 2300 27.5 150 10⁵cop/PCR 25.95 225 23.0 2405 32.3 100 10⁴ cop/PCR 30.2 170 26.6 2350 35.262.5 10³ cop/PCR 34.3 100 30.55 1800 37.55 82.5 10² cop/PCR 36.65 7533.45 1387.5 40.2 52.5 H₂O sample ND ND ND ND ND ND ADN sample ND ND NDND ND ND r2/slope/efficiency PCR 0.988/−3.837/82.2% 0.993/−3.425/95.9%0.976/−3.131/108.6% copy number HPV plasmid/ A9E2AZ8S231f/HPV58A9E2AZ8S231f/HPV67 PCR Moy Ct Moy RFUs Moy Ct Moy RFUs 10⁶ cop/PCR 19.75295 25.45 47.5 10⁵ cop/PCR 22.95 287.5 29.4 47.5 10⁴ cop/PCR 26.4 272.533.1 38.5 10³ cop/PCR 30.35 210 35.8 28.5 10² cop/PCR 33.65 147.5 36.1528.5 H₂O sample ND ND ND ND ADN sample ND ND ND ND r2/slope/efficiencyPCR 0.996/−3.525/92.2% 0.945/−2.778/129.1% ND: not detected NT: nottested

TABLE 83 Megaplex A5 E A6 A A7 A A9 H Kit Kit Quantitect probe PCR MgCl₂5 mM Plasmid concentration 10⁶ cop/PCR Thermoprofile 52° C. Taq 7U/wellCycling 42x forward primer reverse primer probes Name μM Name μM Name μMA5 A5E6f5 0.4 A5E6r5 0.4 A5E6s4 0.2 System E A6 A6E6f1 0.4 A6E6r1 0.4A6E6s1 0.2 System A A7 A7E1-6f1a 0.3 A7E1-6r1b 0.3 A7E1ZCS40f 0.2 SystemA A7E1-6f2a 0.3 A7E1-6r2b 0.3 A7E1ZAS61f 0.2 A7E1-6f3a 0.3 A7E1-6r3b 0.3A7E1ZAS63f 0.2 A7E1ZBS74f 0.2 A9 A9E2f6 0.4 A9E2r10 0.6 A9E2Z8S2f 0.2System H A9E2f8 0.4 A9E2r12B 0.4 A9E2Z8S61f 0.2 A9E2f9 0.6 A9E2r15 0.4A9E2Z8S127f 0.2 A9E2r16 0.4 A9E2Z8S156f 0.2 A9E2Z8S210f 0.2 A9E2Z8S250f0.1

TABLE 84 specificity of megaplex EAAH

ND: no detection NT: not tested

TABLE 85 Megaplex A5 E A6 B A7 A A9 C Kit Kit Quantitect probe PCR MgCl₂5 mM Plasmid concentration 10⁶ cop/PCR Thermoprofile 52° C. Taq 7U/wellCycling 42x Name μM Name μM Name μM A5 System E A5E6f5 0.4 A5E6r5 0.4A5E6s4 0.2 A6 System B A6E6f1 0.6 A6E6r1 0.6 A6E6s1 0.4 A7 A7E1-6f1a 0.5A7E1-6r1b 0.3 A7E1ZCS40f 0.2 System A A7E1-6f2a 0.3 A7E1-6r2b 0.5A7E1ZAS61f 0.2 A7E1-6f3a 0.3 A7E1-6r3b 0.3 A7E1ZAS63f 0.2 A7E1ZBS74f 0.2A9 System C A9E1-f8 0.6 A9E1-r5 0.6 A9E1s10a 0.2 A9E1-f10 0.6 A9E1-r60.6 A9E1s12a 0.2 A9E1-f12 0.2 A9E1-f13 0.4

TABLE 86 specificity of megaplex EBAC

ND: no Detection NT: not tested

TABLE 87 Megaplex A5 E/A6 A/A7 A/A9 H; HPV16 and 18 sensitivity copynumber HPV plasmid/ HPV 16 HPV 18 PCR Moy Ct Moy RFU Moy Ct Moy RFUs 10⁶cop/PCR 25.5 50 25.2 33 10⁵ cop/PCR 28.1 87 27.9 79 10⁴ cop/PCR 31.3106  31.1 72 10³ cop/PCR 35.0 98 34.3 53 10² cop/PCR 37.9 51 37.9 27 H₂Osample ND ND ND ND ADN sample ND ND ND ND r2/slope/efficiency PCR0.989/−3.16/107% 0.989/−3.17/107% ND: not detected NT: not tested

TABLE 88 Megaplex A5 E/A6 B/A7 A/A9 C; HPV16 and 18 sensitivity copynumber HPV plasmid/ HPV 16 HPV 18 PCR Moy Ct Moy RFU Moy Ct Moy RFUs 10⁶cop/PCR 24.3 278 24.3  55 10⁵ cop/PCR 26.8 475 28.1 154 10⁴ cop/PCR 29.9410 31.5 190 10³ cop/PCR 31.2 412 34.3 271 10² cop/PCR 35.8 277 38.0 148H₂O sample ND ND ND ND ADN sample ND ND ND ND r2/slope/efficiency PCR0.984/−2.76/130% 0.995/−3.36/98.5% ND: not detected NT: not tested

TABLE 89 list of HPV sequences Organism Type Accession number human  1 aNC_001356 human  2 a NC_001352 human  3 NC_001588 human  4 NC_001457human  5 NC_001531 human  5 b NC_001444 human  6 NC_000904 human  6 aNC_001668 human  6 b NC_001355 human  7 NC_001595 human  8 NC_001532human  9 NC_001596 human 10 NC_001576 human 11 NC_001525 human 12NC_001577 human 13 NC_001349 human 14 D NC_001578 human 15 NC_001579human 16 AF472509 human 16 NC_001526 human 17 NC_001580 human 18NC_001357 human 19 NC_001581 human 20 NC_001679 human 21 NC_001680 human22 NC_001681 human 23 NC_001682 human 24 NC_001683 human 25 NC_001582human 26 NC_001583 human 27 NC_001584 human 28 NC_001684 human 29NC_001685 human 30 NC_001585 human 31 NC_001527 human 32 NC_001586 human33 NC_001528 human 34 NC_001587 human 35 NC_001529 human 36 NC_001686human 37 NC_001687 human 38 NC_001688 human 39 NC_001535 human 40NC_001589 human 41 NC_001354 human 42 NC_001534 human 44 NC_001689 human45 NC_001590 human 47 NC_001530 human 48 NC_001690 human 49 NC_001591human 50 NC_001691 human 51 NC_001533 human 52 NC_001592 human 53NC_001593 human 54 NC_001676 human 55 NC_001692 human 56 NC_001594 human57 NC_001353 human 57 b HPU37537 human 58 NC_001443 human 59 NC_001635human 60 NC_001693 human 61 NC_001694 human 63 NC_001458 human 65NC_001459 human 66 NC_001695 human 67 D21208 human 68 M73258 human 69NC_002171 human 70 NC_001711 human 71 NC_002644 human 72 X94164 partialE6; 7; 1; 2; 4; L2; 1 human 73 X94165 partial E6; 7; 1; 2; 4; L2; 1human 74 NC_004501 human 82 NC_002172 human 83 NC_000856 human 84NC_002676 human 85 AF131950 human 86 NC_003115 human 87 NC_002627 human89 NC_004103 human 90 NC_004104 human 91 NC_004085 human 92 NC_004500bovine BPV NC_001522 bovine BPV2 NC_001521 bovine BPV3 NC_004197 bovineBPV4 X05817 D00146 X59063 bovine BPV5 NC_004195 canine Canine oralpapillomavirus NC_001619 chimpanzee Common chimpanzee NC_001838papillomavirus rabbit Cottontail rabbit papillomavirus NC_001541 DeerDeer papillomavirus NC_001523 Equinus Equinus papillomavirus NC_004194Equus Equus caballus papillomavirus NC_003748 type 1 elk European elkpapillomavirus NC_001524 Felis Felis domesticus papillomavirus AF480454type 1 coelebs Fringilla coelebs papillomavirus NC_004068 HamsterHamster papovavirus NC_001663 Monkey Monkey B-lymphotropic NC_001536papovavirus rat Multimammate rat papillomavirus NC_001605 Ovine Ovinepapillomavirus 2 NC_001790 Ovine Ovine papillomavirus 1 NC_001789Phocoena Phocoena spinipinnis NC_003348 papillomavirus PsittacusPsittacus erithacus papillomavirus NC_003973 Chimpanzee Pygmy Chimpanzeepapilloma X62844 S43934 virus type 1 Rabbit Rabbit oral papillomavirusNC_002232 Reindeer Reindeer papillomavirus NC_004196

Sequences of the Reference Template Sequences:

<SEQ25; DNA; Human papillomavirus> tggaccgggtcatgttt ggggtgctggagacaaacat ctagagaacc tagagaatct acagtataat catgcatggt aaagtaccaacgctgcaaga cgt <SEQ334; DNA; Human papillomavirus> tggaccgggtcatgtttggggtgctgg agacaaacat ctagagaacc tagagaatct acagtataat catgcatggtaaagtaccaa cgctgcaaga cgttgtatta gaactaacac ctcaaacaga aattgacctacagtgcaatg agcaattgga cagctcagag gatgaggatg aggatgaagt agaccatttgcaggagcggc cacagcaagc tagacaagct aaacaacata cgtgttacct aatacacgtaccttgttgtg agtgtaagtt tgtggtgcag t <SEQ26; DNA; Human papillomavirus>ctaatagcacat ggttggaccg ggtcatgttt ggggtgctgg agacaaacat ctagagaacctagagaatct acagtataat catgcatggt aaagtaccaa cgctgcaaga cgt <SEQ335; DNA;Human papillomavirus> ctaatagcacat ggttggaccg ggtcatgttt ggggtgctggagacaaacat ctagagaacc tagagaatct acagtataat catgcatggt aaagtaccaacgctgcaaga cgttgtatta gaactaacac ctcaaacaga aattgaccta cagtgcaatgagcaattgga cagctcagag gatgaggatg aggatgaagt agaccatttg caggagcggccacagcaagc tagacaagct aaacaacata cgtgttacct aatacacgta ccttgttgtgagtgtaagtt tgtggtgcag t <SEQ27;DNA; Human papillomavirus>aaggtgctacagatgtca aagtccgtta actccggagg aaaagcaatt gcattgtgacagaaaaagac gatttcatct aatagcacat ggttggaccg ggtcatgttt ggggtgctggagacaaacat ctagagaacc <SEQ336; DNA; Human papillomavirus>aaggtgctacagatgtca aagtccgtta actccggagg aaaagcaatt gcattgtgacagaaaaagac gatttcatct aatagcacat ggttggaccg ggtcatgttt ggggtgctggagacaaacat ctagagaacc tagagaatct acagtataat catgcatggt aaagtaccaacgctgcaaga cgt <SEQ337; DNA; Human papillomavirus> aaggtgctacagatgtcaaagtccgtta actccggagg aaaagcaatt gcattgtgac agaaaaagac gatttcatctaatagcacat ggttggaccg ggtcatgttt ggggtgctgg agacaaacat ctagagaacctagagaatct acagtataat catgcatggt aaagtaccaa cgctgcaaga cgttgtattagaactaacac ctcaaacaga aattgaccta cagtgcaatg agcaattgga cagctcagaggatgaggatg aggatgaagt agaccatttg caggagcggc cacagcaagc tagacaagctaaacaacata cgtgttacct aatacacgta ccttgttgtg agtgtaagtt tgtggtgcag t<SEQ28; DNA; Human papillomavirus> gttggaccgggtcatgttt ggggtgctggagacaaacat ctagagaacc tagagaatct acagtataat catgcatggt aaagtaccaacgctgcaaga cgt <SEQ338; DNA; Human papillomavirus> gttggaccg ggtcatgtttggggtgctgg agacaaacat ctagagaacc tagagaatct acagtataat catgcatggtaaagtaccaa cgctgcaaga cgttgtatta gaactaacac ctcaaacaga aattgacctacagtgcaatg agcaattgga cagctcagag gatgaggatg aggatgaagt agaccatttgcaggagcggc cacagcaagc tagacaagct aaacaacata cgtgttacct aatacacgtaccttgttgtg agtgtaagtt tgtggtgcag t <SEQ29; DNA; Human papillomavirus>tcagaggatgaggatg aggatgaagt agaccatttg caggagcggc cacagcaagc tagacaagctaaacaacata cgtgttacct aatacacgta ccttgttgtg agtgtaagtt tgtggtgcag t<SEQ1; DNA; Human papillomavirus> ggcagtggaaagcagtgga gacacccttcgcgttgtaca gcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaacaactagcaacg gcgatgg <SEQ320; DNA; Human papillomavirus>ggcagtggaaagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcgaactaagcctggt ttgcccgtgt tgtgcgaaca actagcaacg gcgatggact <SEQ321; DNA;Human papillomavirus> ggcagtggaaagcagtgga gacacccttc gcgttgtacagcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaaca actagcaacggcgatggact gtgaaggtac agaggatgag gg <SEQ2; DNA; Human papillomavirus>agctccgtgttgcag gtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttcgcgttgtaca gcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaacaactagcaacg gcgatggact <SEQ322; DNA; Human papillomavirus>agctccgtgttgcag gtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttcgcgttgtaca gcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaacaactagcaacg gcgatgg <SEQ323; DNA; Human papillomavirus> agctccgtgttgcaggtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttc gcgttgtacagcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaaca actagcaacggcgatggact gtgaaggtac agaggatgag gg <SEQ3; DNA; Human papillomavirus>atatgcgtgacca gctaccagaa ag/acgggctg gacaggctac gtgttacaga attgaagctccgtgttgcag gtgttcaagt gtagtacaac tggcagtgga aagcagtgga gaca <SEQ324;DNA; Human papillomavirus> atatgcgtgacca gctaccagaa agacgggctggacaggctac gtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaactggcagtgga aagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcgaactaagcctggt ttgcccgtgt tgtgcgaaca actagcaacg gcgatgg <SEQ325; DNA; Humanpapillomavirus> atatgcgtgacca gctaccagaa agacgggctg gacaggctacgtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaac tggcagtggaaagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcgaac taagcctggtttgcccgtgt tgtgcgaaca actagcaacg gcgatggact (SEQ326; DNA; Humanpapillomavirus> atatgcgtgacca gctaccagaa agacgggctg gacaggctacgtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaac tggcagtggaaagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcgaac taagcctggtttgcccgtgt tgtgcgaaca actagcaacg gcgatggact gtgaaggtac agaggatgag gg<SEQ327; DNA; Human papillomavirus> atatgcgtgacca gctaccagaa .agacgggctggacaggctac gtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaactggcagtgga aagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcga <SEQ4;DNA; Human papillomavirus> gacaggctacgtgttacaga attgaagctc cgtgttgcaggtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttc gcgttgtacagcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaaca actagcaacggcgatggact gtgaaggtac agaggatgag gg <SEQ328; DNA; Human papillomavirus>gacaggctac gtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaactggcagtgga aagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcgaactaagcctggt ttgcccgtgt tgtgcgaaca actagcaacg gcgatgg <SEQ329; DNA; Humanpapillomavirus> gacaggctac gtgttacaga attgaagctc cgtgttgcag gtgttcaagtgtagtacaac tggcagtgga aagcagtgga gacacccttc gcgttgtaca gcagatgttaatgggcgaac taagcctggt ttgcccgtgt tgtgcgaaca actagcaacg gcgatggact<SEQ330; DNA; Human papillomavirus> gacaggctac gtgttacaga attgaagctccgtgttgcag gtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttcgcgttgtaca gcagatgtta atgggcga <SEQ5; DNA; Human papillomavirus>cgggctggacaggctac gtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaactggcagtgga aagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcga <SEQ331;DNA; Human papillomavirus> cgggctggacaggctac gtgttacaga attgaagctccgtgttgcag gtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttcgcgttgtaca gcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaacaactagcaacg gcgatgg <SEQ332; DNA; Human papillomavirus> cgggctggacaggctacgtgttacaga attgaagctc cgtgttgcag gtgttcaagt gtagtacaac tggcagtggaaagcagtgga gacacccttc gcgttgtaca gcagatgtta atgggcgaac taagcctggtttgcccgtgt tgtgcgaaca actagcaacg gcgatggact <SEQ333; DNA; Humanpapillomavirus> cgggctggacaggctac gtgttacaga attgaagctc cgtgttgcaggtgttcaagt gtagtacaac tggcagtgga aagcagtgga gacacccttc gcgttgtacagcagatgtta atgggcgaac taagcctggt ttgcccgtgt tgtgcgaaca actagcaacggcgatggact gtgaaggtac agaggatgag gg <SEQ122; DNA; Human papillomavirus>ggacgtggtccaga ttaagtttgc acgaggacga ggacaaggaa aacgatggag actctttgccaacgtttaaa tgtgtgtcag gaca <SEQ123; DNA; Human papillomavirus>tagtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat <SEQ124; DNA; Human papillomavirus> tagtaacacta cacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttt <SEQ125; DNA;Human papillomavirus> tagtaacacta cacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat t <SEQ126; DNA; Human papillomavirus>tagtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtc <SEQ127; DNA; Human papillomavirus> tagtaacactacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttg<SEQ128; DNA; Human papillomavirus> agtaacacta cacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat <SEQ129; DNA; Humanpapillomavirus> agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttt <SEQ130; DNA; Human papillomavirus> agtaacactacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat t<SEQ131; DNA; Human papillomavirus> agtaacacta cacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtc <SEQ132; DNA; Humanpapillomavirus> agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttg <SEQ133; DNA; Human papillomavirus>tagtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgacca <SEQ134; DNA; Human papillomavirus> tagtaacacta cacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat tt <SEQ135; DNA;Human papillomavirus> tagtaacacta cacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat <SEQ136; DNA; Human papillomavirus>agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgacca <SEQ137; DNA; Human papillomavirus> agtaacacta cacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat tt <SEQ138; DNA;Human papillomavirus> agtaacacta cacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat <SEQ139; DNA; Human papillomavirus>actacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgacca <SEQ140;DNA; Human papillomavirus> actacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat tt <SEQ141; DNA; Human papillomavirus>actacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat<SEQ142; DNA; Human papillomavirus> acacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgacca <SEQ143; DNA; Human papillomavirus>acacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat tt<SEQ144; DNA; Human papillomavirus> acacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat <SEQ145; DNA; Human papillomavirus>tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgacca <SEQ146;DNA; Human papillomavirus> tacacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat tt <SEQ147; DNA; Human papillomavirus>tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat<SEQ359; DNA; Human papillomavirus> actacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat t <SEQ360; DNA; Human papillomavirus>actacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttt<SEQ361; DNA; Human papillomavirus> actacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttg <SEQ362; DNA; Humanpapillomavirus> actacacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtc <SEQ363; DNA; Human papillomavirus>tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat<SEQ364; DNA; Human papillomavirus> tacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat t <SEQ365; DNA; Human papillomavirus>tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttt<SEQ366; DNA; Human papillomavirus> tacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttg <SEQ367; DNA; Humanpapillomavirus> tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtc <SEQ144; DNA; Human papillomavirus> acacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat <SEQ368; DNA;Human papillomavirus> acacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat t <SEQ369; DNA; Human papillomavirus> acacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttt <SEQ370; DNA;Human papillomavirus> acacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttg <SEQ371; DNA; Human papillomavirus>acacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtc <SEQ372; DNA; Human papillomavirus> t agtaacacta cacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc <SEQ373; DNA; Human papillomavirus> tagtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc t <SEQ374; DNA;Human papillomavirus> t agtaacacta cacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactgga <SEQ375; DNA; Human papillomavirus> t agtaacactacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ376; DNA; Humanpapillomavirus> t agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggatt <SEQ377; DNA; Human papillomavirus> t agtaacacta cacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactggattt <SEQ378; DNA; Humanpapillomavirus> agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc<SEQ379; DNA; Human papillomavirus> agtaacacta cacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc t <SEQ380; DNA; Human papillomavirus> agtaacactacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ381; DNA; Humanpapillomavirus> agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggat <SEQ382; DNA; Human papillomavirus> agtaacacta cacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactggatt <SEQ383; DNA; Humanpapillomavirus> agtaacacta cacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggattt <SEQ384; DNA; Human papillomavirus> actacacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc <SEQ385; DNA; Human papillomavirus> actacacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc t <SEQ386; DNA; Human papillomavirus>actacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ387; DNA; Humanpapillomavirus> actacacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggat <SEQ388; DNA; Human papillomavirus> actacacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggatt <SEQ389; DNA; Human papillomavirus>actacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggattt <SEQ390; DNA;Human papillomavirus> tacacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc<SEQ391; DNA; Human papillomavirus> tacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc t <SEQ392; DNA; Human papillomavirus> tacacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagactta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactgga <SEQ393; DNA; Human papillomavirus>tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ394; DNA; Humanpapillomavirus> tacacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggatt<SEQ395; DNA; Human papillomavirus> tacacccatagt acatttaaaa ggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactggattt <SEQ396; DNA; Human papillomavirus> acacccatagtacatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc <SEQ397; DNA; Human papillomavirus>acacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc t <SEQ398; DNA; Humanpapillomavirus> acacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ399;DNA; Human papillomavirus> acacccatagt acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggat <SEQ400; DNA; Human papillomavirus> acacccatagt acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggatt <SEQ401; DNA; Human papillomavirus>acacccatagt acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggattt <SEQ148; DNA;Human papillomavirus> taaaaggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgacca <SEQ149;DNA; Human papillomavirus> taaaaggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat tt <SEQ150; DNA; Human papillomavirus> taaaaggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat <SEQ151; DNA; Human papillomavirus>acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgacca <SEQ152; DNA; Humanpapillomavirus> acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat tt<SEQ153; DNA; Human papillomavirus> acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat <SEQ154; DNA; Human papillomavirus>ttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgacca <SEQ155; DNA; Humanpapillomavirus> ttaaaaggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat tt<SEQ156; DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat <SEQ402; DNA; Human papillomavirus> acatttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat t <SEQ403; DNA; Humanpapillomavirus> acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttt<SEQ404; DNA; Human papillomavirus> acatttaaaa ggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttg <SEQ405; DNA; Human papillomavirus>acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtc <SEQ406;DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat t <SEQ407; DNA; Human papillomavirus> ttaaaaggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttt <SEQ408; DNA; Human papillomavirus>ttaaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttg <SEQ409; DNA; Humanpapillomavirus> ttaaaaggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtc <SEQ410; DNA; Human papillomavirus> taaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat t <SEQ411; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttt <SEQ412; DNA; Humanpapillomavirus> taaaaggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttg<SEQ413; DNA; Human papillomavirus> taaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtc <SEQ163; DNA; Human papillomavirus>acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc <SEQ162; DNA; Human papillomavirus>acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc t <SEQ164; DNA; Human papillomavirus>acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattgtacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ414; DNA; Humanpapillomavirus> acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ415; DNA; Humanpapillomavirus> acatttaaaa ggtgatgcta atactttaaa atgtttaaga tatagatttaaaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggatt <SEQ161; DNA;Human papillomavirus> acatttaaaa ggtgatgcta atactttaaa atgtttaagatatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggattt<SEQ167; DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc<SEQ165; DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc t<SEQ168; DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactgga <SEQ416; DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacacaatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggat <SEQ417; DNA; Human papillomavirus> ttaaaaggtgatgcta atactttaaaatgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggatt <SEQ165; DNA; Human papillomavirus> ttaaaaggtgatgctaatactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat actgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactggattt <SEQ159; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc <SEQ158; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc t <SEQ160; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactgga <SEQ418; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggat <SEQ419; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtat8ctgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggatt <SEQ157; DNA; Human papillomavirus>taaaaggtgatgcta atactttaaa atgtttaaga tatagattta aaaagcattg tacattgtatactgcagtgt cgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggattt <SEQ169; DNA; Human papillomavirus>gtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactggat <SEQ170; DNA; Human papillomavirus> gtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc t<SEQ171; DNA; Human papillomavirus> gtcgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc <SEQ172; DNA; Humanpapillomavirus> gtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggattt <SEQ173; DNA; Human papillomavirus>gtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgt9accaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactggatt <SEQ174; DNA; Human papillomavirus> gtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactgga <SEQ175; DNA; Human papillomavirus> actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggat <SEQ176; DNA; Human papillomavirus> actgcagtgt cgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc t<SEQ177; DNA; Human papillomavirus> actgcagtgt cgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc <SEQ178; DNA;Human papillomavirus> actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggattt <SEQ179; DNA;Human papillomavirus> actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggatt <SEQ180; DNA;Human papillomavirus> actgcagtgt cgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ181; DNA; Humanpapillomavirus> tgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ182; DNA; Humanpapillomavirus tgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc t <SEQ183; DNA; Human papillomavirus>tgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc <SEQ184; DNA; Human papillomavirus>tgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggattt <SEQ185; DNA; Human papillomavirus>tgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactggatt <SEQ186; DNA; Human papillomavirus>tgcagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaattgttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaataccaaaaacta ttacagtgtc tactgga <SEQ187; DNA; Human papillomavirus>agtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactggat <SEQ188; DNA; Human papillomavirus> agtgtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc t<SEQ189; DNA; Human papillomavirus> agtgtcgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc <SEQ190; DNA;Human papillomavirus> agtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactggattt <SEQ191;DNA; Humanpapillomavirus> agtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactggatt <SEQ192; DNA; Humanpapillomavirus> agtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ193; DNA; Humanpapillomavirus> cagtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ194; DNA; Humanpapillomavirus> cagtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc t <SEQ195; DNA; Human papillomavirus>cagtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc <SEQ196; DNA; Human papillomavirus> cagtgtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggattt <SEQ197; DNA; Human papillomavirus> cagtgtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggatt <SEQ198; DNA; Human papillomavirus> cagtgtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactgga <SEQ199; DNA; Human papillomavirus> tgtcgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ200;DNA; Human papillomavirus> tgtcgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc t <SEQ201; DNA; Humanpapillomavirus> tgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc <SEQ202; DNA; Human papillomavirus>tgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc tactggattt <SEQ203; DNA; Human papillomavirus> tgtcgtctacatggcattggaca ggacataatg taaaacataa aagtgcaatt gttacactta catatgatagtgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtctactggatt <SEQ204; DNA; Human papillomavirus> tgtcgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ205;DNA; Human papillomavirus> gtgtcgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggat <SEQ206; DNA; Humanpapillomavirus> gtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc t <SEQ207; DNA; Human papillomavirus>gtgtcgtctacatg gcattggaca ggacataatg taaaacataa aagtgcaatt gttacacttacatatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaaaactattacagtgtc <SEQ208; DNA; Human papillomavirus> gtgtcgtctacatg gcattggacaggacataatg taaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaacgtgaccaat ttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggattt<SEQ209; DNA; Human papillomavirus> gtgtcgtctacatg gcattggaca ggacataatgtaaaacataa aagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaatttttgtctca agttaaaata ccaaaaacta ttacagtgtc tactggatt <SEQ210; DNA;Human papillomavirus> gtgtcgtctacatg gcattggaca ggacataatg taaaacataaaagtgcaatt gttacactta catatgatag tgaatggcaa cgtgaccaat ttttgtctcaagttaaaata ccaaaaacta ttacagtgtc tactgga <SEQ46; DNA; Humanpapillomavirus> tggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatg <SEQ47; DNA; Humanpapillomavirus> tggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc ag <SEQ48; DNA; Humanpapillomavirus> tggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca atagcaatgc agc <SEQ49; DNA; Humanpapillomavirus> gaacaggaatatcaaatat tagtgaagta atgggagaca cacctgagtggatacaaaga cttactatta tacaacatgg aatagatgat agcaattttg atttgtcagaaatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaatatgccttatta gcagacagca acagcaatg <SEQ50; DNA; Human papillomavirus>gaacaggaatatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaagacttactatta tacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaata tgccttattagcagacagca acagcaatgc ag <SEQ51; DNA; Human papillomavirus>gaacaggaatatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaagacttactatta tacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaata tgccttattagcagacagca acagcaatgc agc <SEQ52; DNA; Human papillomavirus>tggtatagaacaggaa tatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaagacttactatta tacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg ca <SEQ53; DNA; Humanpapillomavirus> tggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatgg cattt<SEQ339; DNA; Human papillomavirus> gaacaggaa tatcaaatat tagtgaagtaatgggagaca cacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgatagcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaaagcgatatgg ca <SEQ340; DNA; Human papillomavirus> gaacaggaatatcaaatattagtgaagta atgggagaca cacctgagtg gatacaaaga cttactatta tacaacatggaatagatgat agcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagctgacagatgaa agcgatatgg cattt <SEQ341; DNA; Human papillomavirus>tggtatagaacaggaa tatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaagacttactatta tacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaata tgccttattagcagacagca acagcaatgc <SEQ342; DNA; Human papillomavirus>gaacaggaatatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaagacttactatta tacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaata tgccttattagcagacagca acagcaatgc <SEQ54; DNA; Human papillomavirus> ggtatagaacaggaatatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaaga cttactattatacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaa tgggcatttgataatgagct gacagatgaa agcgatatgg ca <SEQ55; DNA; Human papillomavirus>ggtatagaacaggaa tatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaagacttactatta tacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg cattt <SEQ56; DNA; Humanpapillomavirus> gtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatgg ca<SEQ57; DNA; Human papillomavirus> gtatagaacaggaa tatcaaatat tagtgaagtaatgggagaca cacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgatagcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaaagcgatatgg cattt <SEQ343; DNA; Human papillomavirus> ggtatagaacaggaatatcaaatat tagtgaagta atgggagaca cacctgagtg gatacaaaga cttactattatacaacatgg aatagatgat agcaattttg atttgtcaga aatggtacaa tgggcatttgataatgagct acagatgaa agcgatatgg catttgaata tgccttatta gcagacagcaacagcaatg <SEQ344; DNA; Human papillomavirus> ggtatagaacaggaa tatcaaatattagtgaagta atgggagaca cacctgagtg gatacaaaga cttactatta tacaacatggaatagatgat agcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagctgacagatgaa agcgatatgg catttgaata tgccttatta gcagacagca acagcaatgc ag<SEQ345; DNA; Human papillomavirus> ggtatagaacaggaa tatcaaatattagtgaagta atgggagaca cacctgagtg gatacaaaga cttactatta tacaacatggaatagatgat agcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagctgacagatgaa agcgatatgg catttgaata tgccttatta gcagacagca acagcaatgc agc<SE0346; DNA; Human papillomavirus> gtatagaacaggaa tatcaaatat tagtgaagtaatgggagaca cacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgatagcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaaagcgatatgg catttgaata tgccttatta gcagacagca acagcaatg <SEQ347; DNA;Human papillomavirus> gtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc ag <SEQ348; DNA; Humanpapillomavirus> gtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc agc <SEQ349; DNA; Humanpapillomavirus> tggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc <SEQ350; DNA; Humanpapillomavirus> ggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc <SEQ345; DNA; Humanpapillomavirus> ggtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc agc <SEQ351; DNA; Humanpapillomavirus> gtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga ettactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc <SEQ352; DNA; Humanpapillomavirus> gtatagaacaggaa tatcaaatat tagtgaagta atgggagacacacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat agcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc agc <SEQ58; DNA; Humanpapillomavirus> tgatagcaattttg atttgtcaga aatggtacaa tgggcatttgataatgagct gacagatgaa agcgatatgg catttgaata tgccttatta gcagacagcaacagcaatgc <SEQ59; DNA; Human papillomavirus> tgatagcaattttg atttgtcagaaatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaatatgccttatta gcagacagca acagcaatgc agc <SEQ60; DNA; Human papillomavirus>gatagcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaaagcgatatgg catttgaata tgccttatta gcagacagca acagcaatgc <SEQ61; DNA;Human papillomavirus> gatagcaattttg atttgtcaga aatggtacaa tgggcatttgataatgagct gacagatgaa agcgatatgg catttgaata tgccttatta gcagacagcaacagcaatgc agc <SEQ62; DNA; Human papillomavirus> ggaatagatgatagcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaaagcgatatgg catttgaata tgccttatta gcagacagca acagcaatgc <SEQ63; DNA;Human papillomavirus> ggaatagatgat agcaattttg atttgtcaga aatggtacaatgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaata tgccttattagcagacagca acagcaatgc agc <SEQ353; DNA; Human papillomavirus>ggaatagatgat agcaattttg atttgtcaga aatggtacaa tgggcatttg taatgagctgacagatgaa agcgatatgg catttgaata tgccttatta gcagacagca acagcaatg<SEQ354; DNA; Human papillomavirus> ggaatagatgat agcaattttg atttgtcagaaatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaatatgccttatta gcagacagca acagcaatgc ag <SEQ355; DNA; Human papillomavirus>tgatagcaattttg atttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaaagcgatatgg catttgaata tgccttatta gcagacagca acagcaatg <SEQ356; DNA;Human papillomavirus> tgatagcaattttg atttgtcaga aatggtacaa tgggcatttgataatgagct gacagatgaa agcgatatgg catttgaata tgccttatta gcagacagcaacagcaatgc ag <SEQ59; DNA; Human papillomavirus> tgatagcaattttgatttgtcaga aatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatggcatttgaata tgccttatta gcagacagca acagcaatgc agc <SEQ357; DNA; Humanpapillomavirus> gatagcaattttg atttgtcaga aatggtacaa tgggcatttgataatgagct gacagatgaa agcgatatgg catttgaata tgccttatta gcagacagcaacagcaatg <SEQ358; DNA; Human papillomavirus> gatagcaattttg atttgtcagaaatggtacaa tgggcatttg ataatgagct gacagatgaa agcgatatgg catttgaatatgccttatta gcagacagca acagcaatgc ag <SEQ64; DNA; Human papillomavirus>ggctgatccagaagg tacagacggg gagggcacgg gttgtaacgg ctggttttat gtacaagctattgtagacaa aaaaacagga gatgtaatat cagatgacga ggacgaaaat gc <SEQ65; DNA;Human papillomavirus> ggctgatccagaagg tacagacggg gagggcacgg gttgtaacggctggttttat gtacaagcta ttgtagacaa aaaaacagga gatgtaatat cagatgacgaggacgaaaat gcaacagaca cagg <SEQ66; DNA; Human papillomavirus>gatccagaagg tacagacggg gagggcacgg gttgtaacgg ctggttttat gtacaagctattgtagacaa aaaaacagga gatgtaatat cagatgacga ggacgaaaat gc <SEQ67; DNA;Human papillomavirus> gatccagaagg tacagacggg gagggcacgg gttgtaacggctggttttat gtacaagcta ttgtagacaa aaaaacagga gatgtaatat cagatgacgaggacgaaaat gcaacagaca cagg

1. Process for detecting in a sample at least one HPV, which can beoncogenic for the mucosal epithelia, wherein said detection comprisesthe determination of whether at least one amplicon has been, or is,produced from said sample, or from nucleic acid material thereof, byamplification by means of amplification primers, whereby the productionof at least one amplicon indicates that at least one HPV, which can beoncogenic for the mucosal epithelia, is present in said sample,characterized in that said amplification primers comprise: at least twoprimers, which are intended for targeting oncogenic HPV of group A6,wherein said at least two A6-targeted primers are oligonucleotides,which consist of 14-30 nucleotides, preferably of 17-25 nucleotides, thesequences of which are suitable for use as forward and reverse primers,respectively, in the amplification of at least one A6 reference templatesequence, wherein said at least one A6 reference template sequence is afragment consisting of positions 413-791 (SEQ ID NO:337) of the HPV56sequence of SEQ ID NO:420 (accession NC_(—)001594.1), or of aconservative sub-fragment thereof, which has retained the property ofbeing a suitable reference template sequence, to construct and produceA6-targeted primers, which allow for a real-time multiplex detection ofthose HPV, which can be oncogenic for the mucosal epithelia, and/or atleast two primers, which are intended for targeting oncogenic HPV ofgroup A5, wherein said at least two A5-targeted primers areoligonucleotides, which consist of 14-30 nucleotides, preferably of17-25 nucleotides, the sequences of which are suitable for use asforward and reverse primers, respectively, in the amplification of atleast one A5 reference template sequence, which is a fragment consistingof positions 678-902 (SEQ ID NO:326) of the HPV51 sequence of SEQ IDNO:421 (accession NC_(—)001533.1), or of a conservative sub-fragmentthereof, which has retained the property of being a suitable referencetemplate sequence, to construct and produce A5-targeted primers, whichallow for a real-time multiplex detection of those HPV, which can beoncogenic for the mucosal epithelia,  and/or at least two primers, whichare intended for targeting oncogenic HPV of group A9, wherein said atleast two A9-targeted primers are oligonucleotides, which consist of14-30 nucleotides, preferably of 17-25 nucleotides, the sequences ofwhich are suitable for use as forward and reverse primers, respectively,in the amplification of at least one A9 reference template sequence,which is: a fragment consisting of positions 2707-2794 (SEQ ID NO:122)of the HPV16 sequence of SEQ ID NO:422 (accession NC_(—)001526.1), or aconservative sub-fragment thereof, which has retained the property ofbeing a suitable reference template sequence, to construct and produceA9-targeted primers, which allow for a real-time multiplex detection ofthose HPV, which can be oncogenic for the mucosal epithelia, or afragment consisting of positions 3600-3840 (SEQ ID NO:377) of the HPV16sequence of SEQ ID NO:422 (accession NC_(—)001526.1), or of aconservative sub-fragment thereof, which has retained the property ofbeing a suitable reference template sequence, to construct and produceA9-targeted primers, which allow for a real-time multiplex detection ofthose HPV, which can be oncogenic for the mucosal epithelia, and/or atleast two primers, which are intended for targeting oncogenic HPV ofgroup A7, wherein said at least two A7-targeted primers areoligonucleotides, which consist of 14-30 nucleotides, preferably of17-25 nucleotides, the sequences of which are suitable for use asforward and reverse primers, respectively, in the amplification of atleast one A7 reference template sequence, wherein said at least onereference template sequence is: a fragment consisting of positions1895-2103 (SEQ ID NO:48) of the HPV18 sequence of SEQ ID NO:423(accession NC_(—)001357.1), or of a conservative sub-fragment thereof,which has retained the property of being a suitable reference templatesequence, to construct and produce A7-targeted primers, which allow fora real-time multiplex detection of those HPV, which can be oncogenic forthe mucosal epithelia, or a fragment consisting of positions 916-1044(SEQ ID NO:65) of the HPV18 sequence of SEQ ID NO:423 (accessionNC_(—)001357.1), or of a conservative sub-fragment thereof, which hasretained the property of being a suitable reference template sequence,to construct and produce A7-targeted primers, which allow for areal-time multiplex detection of those HPV, which can be oncogenic forthe mucosal epithelia, said A6, A5, A9 and A7 reference templatesequences sharing the special technical feature of being group-basedreference template sequences, which are suitable to construct andproduce primers and amplicon-annealing probes, which allow for areal-time multiplex amplification of at least the five most common HRHPV (HPV16, 18, 45, 31, 33), preferably of at least 7 HR HPV, stillpreferably of the five most common HR HPV as well as at least two otherHR HPV, advantageously at least two other HR HPV belonging to groups A6and/or A5 (e.g., HPV 56, 51, 33, 31, 16, 45, 18), more preferably of atleast the 13 HR HPV (HPV56, 51, 58, 33, 52, 35, 31, 16, 68, 39, 59, 45and 18), and more particularly for a real-time quantitative multiplexamplification of such HPV.
 2. HPV detection process according to claim1, characterized in that said at least one A6 reference templatesequence is one of SEQ ID NO:25-29 and NO:334-338, said A6 referencetemplate sequences sharing the specific technical feature of beingsuitable references to construct and produce A6-targeted primers, whichallow for a real-time multiplex amplification of HPV, which can beoncogenic for the mucosal epithelia.
 3. HPV detection process accordingto claim 1, characterized in that said at least one A5 referencetemplate sequence is one of SEQ ID NO: 1-5 and NO:320-333, said A5reference template sequences sharing the specific technical feature ofbeing suitable references to construct and produce A5-targeted primers,which allow for a real-time multiplex amplification of HPV, which can beoncogenic for the mucosal epithelia.
 4. HPV detection process accordingto claim 1, characterized in that said at least one A9 referencetemplate sequence is one of SEQ ID NO:122-210 and 359-419, said A9reference template sequences sharing the specific technical feature ofbeing suitable references to construct and produce A9-targeted primers,which allow for a real-time multiplex amplification of HPV, which can beoncogenic for the mucosal epithelia.
 5. HPV detection process accordingto claim 1 characterized in that said at least one A7 reference templatesequence is one of one of SEQ ID NO:46-67; 339-358, said A7 referencetemplate sequences sharing the specific technical feature of beingsuitable references to construct and produce A7-targeted primers, whichallow for a real-time multiplex amplification of HPV, which can beoncogenic for the mucosal epithelia.
 6. HPV detection process accordingto claim 1, characterized in that: said at least two A9-targeted primersare oligonucleotides, which consist of 14-30 nucleotides, the sequencesof which are suitable for use as forward and reverse primers,respectively, in the amplification of at least one nucleic acid of atleast 80-260 nucleotides, preferably of 85-250 nucleotides, morepreferably of 88-241 nucleotides from the A9-target region consisting ofthe E1 and E2 genes from each of the following group A9 HPV: HPV58,HPV33, HPV52, HPV35, HPV31, and/or said at least two A7-targeted primersare oligonucleotides, which consist of 14-30 nucleotides, the sequencesof which are suitable for use as forward and reverse primers,respectively, in the amplification of at least one nucleic acid of atleast 100-220 nucleotides, preferably of 120-215 nucleotides, morepreferably of 125-209 nucleotides from the A7-target region consistingof the E1 gene from each of the following group A7 HPV: HPV68, HPV39,HPV59, HPV45.
 7. HPV detection process according to claim 6,characterized in that: said at least two A6-targeted primers are furthersuitable for use as forward and reverse primers, respectively, in theamplification of at least one nucleic acid of 90-390 nucleotides,preferably of 95-385 nucleotides, more preferably of 100-379 nucleotidesfrom the A6-target region consisting of the E6 and E7 genes of HPV66and/or HPV53, and/or said at least two A5-targeted primers are furthersuitable for use as forward and reverse primers, respectively, in theamplification of at least one nucleic acid of 90-240 nucleotides,preferably of 100-230 nucleotides, more preferably of 106-225nucleotides from the A5-target region consisting of the E7 and E1 genesof HPV82, and/or said at least two A9-targeted primers are furthersuitable for use as forward and reverse primers, respectively, in theamplification of at least one nucleic acid of 80-260 nucleotides,preferably of 85-250 nucleotides, more preferably of 88-241 nucleotidesfrom the A9-target region consisting of the E1 and E2 genes of HPV67,and/or said at least two A7-targeted primers are further suitable foruse as forward and reverse primers, respectively, in the amplificationof at least one nucleic acid of 100-220 nucleotides, preferably of120-215 nucleotides, more preferably of 125-209 nucleotides from theA7-target region consisting of the E1 and E2 genes of HPV85. 8.(canceled)
 9. HPV detection process according claim 1, characterized inthat said at least one HPV, which can be oncogenic for the mucosalepithelia is an oncogenic anogenital HPV or an oncogenic cervical HPV.10. (canceled)
 11. HPV detection process according to claim 1,characterized in that said at least two primers are specific of groupA6, or group A5, or group A9, or group A7.
 12. (canceled)
 13. HPVdetection process according to claim 1, characterized in that saidamplification primers comprise at least two elements from the followinglist of four elements. Preferably the following four elements: said atleast two A6-targeted primers, said at least two A5-targeted primers,said at least two A9-targeted primers, said at least two A7-targetedprimers. 14-18. (canceled)
 19. HPV detection process according to claim1, characterized in that said amplification is a real-timeamplification.
 20. (canceled)
 21. HPV detection process according toclaim 1, characterized in that said amplification is a real-timemultiplex amplification.
 22. HPV detection process according to claim 1,characterized in that said amplification is a quantitative real-timemultiplex amplification.
 23. HPV detection process according to claim 1,characterized in that said at least two A6-targeted primers are at leastone of SEQ ID NO: 30-34 (forward primer) and at least one of SEQ ID NO:35-37 (reverse primer).
 24. HPV detection process according to claim 1,characterized in that said determination of whether at least oneamplicon is produced is carried out by using in real-time amplificationat least one A6-targeted probe, which consists of one of SEQ IDNO:38-40, or of one of the complementary sequences thereof, andoptionally at least one 5′ and/or 3′ detection label and/or at least oneHPV-unrelated arm intended to carry a quencher or a reporter.
 25. HPVdetection process according to claim 24, characterized in that said atleast one A6-targeted probe is a beacon probe, the sequence of which isone of SEQ ID NO:41-45, or of one of the complementary sequencesthereof.
 26. HPV detection process according to claim 1, characterizedin that said at least two A5-targeted primers are at least one of SEQ IDNO: 6-10 (forward primer) and at least one of SEQ ID NO: 11-15 (reverseprimer).
 27. HPV detection process according to claim 1, characterizedin that said determination of whether at least one amplicon is producedis carried out by using in real-time amplification at least oneA5-targeted probe, which consists of one of SEQ ID NO:16-19, or of oneof the complementary sequences thereof, and optionally at least onedetection label and/or at least one HPV-unrelated arm intended to carrya quencher or a reporter.
 28. HPV detection process according to claim27, characterized in that said at least one A5-targeted probe is abeacon probe, the sequence of which is one of SEQ ID NO: 20-24, or ofone of the complementary sequences thereof.
 29. HPV detection processaccording to claim 1, characterized in that said at least twoA9-targeted primers are at least one of SEQ ID NO: 211-239 (forwardprimers) and at least one of SEQ ID NO: 240-265 (reverse primers). 30.HPV detection process according to claim 1, characterized in that saiddetermination of whether at least one amplicon is produced is carriedout by using in real-time amplification at least one A9-targeted probe,which consists of one of SEQ ID NO: 266-282, or of one of thecomplementary sequences thereof, and optionally at least one detectionlabel and/or at least one HPV-unrelated arm intended to carry a quencheror a reporter.
 31. HPV detection process according to claim 30,characterized in that said at least one A9-targeted probe is a beaconprobe, the sequence of which is one of SEQ ID NO: 283-319, or of one ofthe complementary sequences thereof.
 32. HPV detection process accordingto claim 1, characterized in that said at least two A7-targeted primersare at least one of SEQ ID NO: 68-78 (forward primer) and at least oneof SEQ ID NO: 79-87 (reverse primer).
 33. HPV detection processaccording to claim 1, characterized in that said determination ofwhether at least one amplicon is produced is carried out by using inreal-time amplification at least one A7-targeted probe, which consistsof one of SEQ ID NO:88-101, or of one of the complementary sequencesthereof, and optionally at least one detection label and/or at least oneHPV-unrelated arm intended to carry a quencher or a reporter.
 34. HPVdetection process according to claim 33, characterized in that said atleast one A7-targeted probe is a beacon probe, the sequence of which isone of SEQ ID NO:102-121, or of one of the complementary sequencesthereof.
 35. (canceled)
 36. Amplicon obtainable by implementation of theprocess according to claim 1 on a HPV-containing sample, which containsat least one HPV of group A6, A5, A9 or A7.
 37. Polynucleotide suitablefor use as a reference template sequence in the design of primers thatcan be used in a single-tube multiplex to amplify those HPV of groupsA6, A5, A9 and A7, and in the design of probes that can be used in saidsingle-tube multiplex for real-time detection of said amplified HPV,said reference template polynucleotide being selected from: for groupA6: a fragment consisting of positions 413-791 (SEQ ID NO:337) of theHPV56 sequence of SEQ ID NO:420 (accession NC_(—)001594.1), or of aconservative sub-fragment thereof, or a sequence which is fullycomplementary to said fragment or sub-fragment over the entire length ofsaid fragment or sub-fragment; for group A5: a fragment consisting ofpositions 678-902 (SEQ ID NO:326) of the HPV51 sequence of SEQ ID NO:421(accession NC_(—)001533.1), or of a conservative sub-fragment thereof,or a sequence which is fully complementary to said fragment orsub-fragment over the entire length of said fragment or sub-fragment;for group A9: a fragment consisting of positions 2707-2794 (SEQ IDNO:122) of the HPV16 sequence of SEQ ID NO:422 (accessionNC_(—)001526.1), or a conservative sub-fragment thereof, or a sequencewhich is fully complementary to said fragment or sub-fragment over theentire length of said fragment or sub-fragment; or a fragment consistingof positions 3600-3840 (SEQ ID NO:377) of the HPV16 sequence of SEQ IDNO:422 (accession NC_(—)001526.1), or of a conservative sub-fragmentthereof, or a sequence which is fully complementary to said fragment orsub-fragment over the entire length of said fragment or sub-fragment;for group A7: a fragment consisting of positions 1895-2103 (SEQ IDNO:48) of the HPV18 sequence of SEQ ID NO:423 (accessionNC_(—)001357.1), or of a conservative sub-fragment thereof, or asequence which is fully complementary to said fragment or sub-fragmentover the entire length of said fragment or sub-fragment; or a fragmentconsisting of positions 916-1044 (SEQ ID NO:65) of the HPV18 sequence ofSEQ ID NO:423 (accession NC_(—)001357.1), or of a conservativesub-fragment thereof, or a sequence which is fully complementary to saidfragment or sub-fragment over the entire length of said fragment orsub-fragment; wherein said conservative fragment thereof have retainedthe property of being a suitable reference template sequence, toconstruct and produce group-targeted primers, which allow for areal-time multiplex detection of those HPV, which can be oncogenic forthe mucosal epithelia.
 38. Reference template polynucleotide accordingto claim 37, characterized in that it is a group A6-targeted referencetemplate sequence, and in that it consists of one of SEQ ID NO:25-29 andNO:334-338, or a sequence which is fully complementary thereto over theentire length of said SEQ ID NO sequence.
 39. Reference templatepolynucleotide according to claim 37, characterized in that it is agroup A5-targeted reference template sequence, and in that it consistsof one of SEQ ID NO: 1-5 and NO:320-333, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence.40. Reference template polynucleotide according to claim 37,characterized in that it is a group A9-targeted reference templatesequence, and in that it consists of one of SEQ ID NO:122-210 and359-419, or a sequence which is fully complementary thereto over theentire length of said SEQ ID NO sequence.
 41. Reference templatepolynucleotide according to claim 37, characterized in that it is agroup A7-targeted reference template sequence, and in that it consistsof one of SEQ ID NO: 46-67; 339-358, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence.42. Primer suitable for HPV amplification, which is especially adaptedto the real-time multiplex amplification of HPV groups A6 and A5 and A9and A7, said primer being: an A6-targeted primer, consisting of any oneof SEQ ID NO: 30-34 and SEQ ID NO: 35-37; or an A5-targeted primer,consisting of any one of SEQ ID NO: 6-10 and SEQ ID NO: 11-15; or anA9-targeted primer, consisting of any one of SEQ ID NO: 211-239 and SEQID NO: 240-265; or an A7-targeted primer, consisting of any one of SEQID NO: 68-78 and SEQ ID NO: 79-87.
 43. Primer system suitable for HPVamplification, which is especially adapted to the real-time multiplexamplification of HPV groups A6 and A5 and A9 and A7, said primer systemcomprising: at least one A6-targeted primer consisting of one of SEQ IDNO: 30-34, and at least one A6-targeted primer consisting of one of SEQID NO: 35-37; and/or at least one A5-targeted primer consisting of oneof SEQ ID NO: 6-10, and at least one A5-targeted primer consisting ofone of SEQ ID NO: 11-15; and/or at least one A9-targeted primerconsisting of one of SEQ ID NO: 211-239, and at least one A9-targetedprimer consisting of one of SEQ ID NO: 240-265; and/or at least oneA7-targeted primer consisting of one of SEQ ID NO: 68-78, and at leastone A7-targeted primer consisting of one of SEQ ID NO: 79-87.
 44. Probesuitable for HPV detection, which is especially adapted to the real-timemultiplex amplification of HPV groups A6 and A5 and A9 and A7, saidprobe being: an A6-targeted probe, consisting of any one of SEQ IDNO:38-40, or a sequence which is fully complementary thereto over theentire length of said SEQ ID NO sequence; or an A5-targeted probe,consisting of any one of SEQ ID NO:16-19, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence;or an A9-targeted probe, consisting of any one of SEQ ID NO: 266-282, ora sequence which is fully complementary thereto over the entire lengthof said SEQ ID NO sequence; or an A7-targeted probe, consisting of anyone of SEQ ID NO:88-101, or a sequence which is fully complementarythereto over the entire length of said SEQ ID NO sequence.
 45. Beaconprobe suitable for HPV detection, which is especially adapted to thereal-time multiplex amplification of HPV groups A6 and A5 and A9 and A7,said beacon probe being: an A6-targeted probe, consisting of any one ofSEQ ID NO:41-45, or a sequence which is fully complementary thereto overthe entire length of said SEQ ID NO sequence; or an A5-targeted probe,consisting of any one of SEQ ID NO:20-24, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence;or an A9-targeted probe, consisting of any one of SEQ ID NO: 283-319, ora sequence which is fully complementary thereto over the entire lengthof said SEQ ID NO sequence; or an A7-targeted probe, consisting of anyone of SEQ ID NO:102-121, or a sequence which is fully complementarythereto over the entire length of said SEQ ID NO sequence.
 46. Primerand probe system, which is suitable for HPV amplification, and which isespecially adapted to the real-time multiplex amplification of HPVgroups A6 and A5 and A9 and A7, said primer and probe system comprisingat least one primer system according to claim 43, and at least one probesuitable for HPV detection, which is especially adapted to the real-timemultiplex amplification of HPV groups A6 and A5 and A9 and A7, saidprobe being: an A6-targeted probe, consisting of any one of SEQ IDNO:38-40, or a sequence which is fully complementary thereto over theentire length of said SEQ ID NO sequence; or an A5-targeted probe,consisting of any one of SEQ ID NO:16-19, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence;or an A9-targeted probe, consisting of any one of SEQ ID NO: 266-282, ora sequence which is fully complementary thereto over the entire lengthof said SEQ ID NO sequence; or an A7-targeted probe, consisting of anyone of SEQ ID NO:88-101, or a sequence which is fully complementarythereto over the entire length of said SEQ ID NO sequence; or a beaconprobe suitable for HPV detection, which is especially adapted to thereal-time multiplex amplification of HPV groups A6 and A5 and A9 and A7,said beacon probe being: an A6-targeted probe, consisting of any one ofSEQ ID NO:41-45, or a sequence which is fully complementary thereto overthe entire length of said SEQ ID NO sequence; or an A5-targeted probe,consisting of any one of SEQ ID NO:20-24, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence;or an A9-targeted probe, consisting of any one of SEQ ID NO: 283-319, ora sequence which is fully complementary thereto over the entire lengthof said SEQ ID NO sequence; or an A7-targeted probe, consisting of anyone of SEQ ID NO:102-121, or a sequence which is fully complementarythereto over the entire length of said SEQ ID NO sequence.
 47. Ampliconobtainable by amplification of at least one nucleic acid from an HPV ofgroup A6, A5, A9 or A7, by means of at least one primer system accordingto claim
 43. 48. Amplification composition comprising at least oneamplicon according to claim
 36. 49. Kit for the diagnostic or prognosticof a cervical neoplasia or cancer, comprising: at least one primersystem according to claim 43, and/or at least one probe suitable for HPVdetection, which is especially adapted to the real-time multiplexamplification of HPV groups A6 and A5 and A9 and A7, said probe being:an A6-targeted probe, consisting of any one of SEQ ID NO:38-40, or asequence which is fully complementary thereto over the entire length ofsaid SEQ ID NO sequence; or an A5-targeted probe, consisting of any oneof SEQ ID NO:16-19, or a sequence which is fully complementary theretoover the entire length of said SEQ ID NO sequence; or an A9-targetedprobe, consisting of any one of SEQ ID NO: 266-282, or a sequence whichis fully complementary thereto over the entire length of said SEQ ID NOsequence; or an A7-targeted probe, consisting of any one of SEQ IDNO:88-101, or a sequence which is fully complementary thereto over theentire length of said SEQ ID NO sequence; or a beacon probe suitable forHPV detection, which is especially adapted to the real-time multiplexamplification of HPV groups A6 and A5 and A9 and A7, said beacon probebeing: an A6-targeted probe, consisting of any one of SEQ ID NO:41-45,or a sequence which is fully complementary thereto over the entirelength of said SEQ ID NO sequence; or an A5-targeted probe, consistingof any one of SEQ ID NO:20-24, or a sequence which is fullycomplementary thereto over the entire length of said SEQ ID NO sequence;or an A9-targeted probe, consisting of any one of SEQ ID NO: 283-319, ora sequence which is fully complementary thereto over the entire lengthof said SEQ ID NO sequence; or an A7-targeted probe, consisting of anyone of SEQ ID NO:102-121, or a sequence which is fully complementarythereto over the entire length of said SEQ ID NO sequence optionally,instructions for the use thereof and/or nucleotides.